Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~U4Z~6()
The present invention relates to a novel substituted
butanoic ~cid and to methods of preparing this material. More
particularly, the present invention relates to the new substance
2-amino-4-12-aminoethoxy)-butanoic acid~ i.e, the compound of
the formula I
~U2 I
H2NCH2CH20CH2CH2CHCOOH
and the biologically acceptable acid addition salts thereof.
The I,antipode of the compound of formula I is preferred.
The process ~r the preparation of compounds of
formula I comprises
a) reducing a compound of formula II
H2NCH2CH20~ f ~ . '
/C==C\ ' ~ ~
H CH_COOH
1H2 II
in the presence of a catalyst, or
b) condensing a compound of the formula III
RlHN2CH2CH2-O--c~-cH2R2 III
: i
'. ~
-2-
wherein Rl signifies a suitable nitrogen protecting
group and R2 signifies chlorine, bromine or iodine,
with the alkalimetal derivative of a compound of the formula IV
COOC2H5
HF-NHR3 IV ~
COOC2H5 "' ~ " "
wherein R3 signifies a suitable nitrogen protecting
group,
and removing any nitrogen protecting group present in a convention-
al manner, or
c) resolving the racemate of a compound of formula I and
isolating the L-antipode in a conventional manner, or
d) converting a compound of formula I which is basic in
nature into a ~ acceptable acid addition
salt.
In a specific process aspect, the compound of formula
I above can be prepared e.g. by the catalytic reduction o~ the
known compound L-trans-2-amino-4-(2-aminoethoxy)-3-butanoic ~ ;
acid, i.e. the L-antipode of the compound of the formula:
' ~.
'
~ ' ,
- 3 -
:,., ,, - ; , : ~ . : . , .. ,, .. . , :
~4Z~6~
H2~cH2cH2 \ H
C ---C
H \ ~H-COOH II
NH2
The compound of formula II above is known; its preparation is
described in United States patent 3,751,459 issued August 7, 1973 in the
names of Berger, Pruess and Scannell.
Conversion of the compo~d of formula II above to the desired
product of formula I is accomplished by catalytic reduction. Suitable
reducing systems for this purpose include hydrogen in the presnece of 5%
palladium on charcoal or hydrogen in the presence of 10% platinum on
charcoal. This catalytic reduction is expediently effected in the presence
of an inert solvent such as water, lower alkanols such as methanol, e~hanol
and the like or mixtures of water and a lower alkanol. Temperature and
pressure are not critical to this process aspect and thus the reaction
may be performed at room temperature or above~ with room temperature and
atmospheric pressure being the preferred conditions.
In a further process aspect the desired compound of formula I
above may be prepared by the resolution of the corresponding racemic
compound, D~L-2-amino-4-(2-aminoethoxy)-butanoic acid. This racemic
compound is novel and as such forms a part of the present invention. It
can be prepared by condensing a compound of the formula
~HN-CH2CH2-0-CH2CH2R2 III
wherein ~ signifies a suitable nitrogen protecting
group and R2 signifies chlorine, bromine or iodine
with the alkali metal derivative of a compound of the formula
COOC2H5
f 3 IV
C2H5 ;~
-4-
-" :
. ;. , . , . . ~ . . : :.
~04246~
wherein R3 signifies a suitable nitrogen protecting
group.
The alkali metal derivative of the compound of formula IV above
may be prepared following conventional techniques, such as by treating
said compound with an alkali metal alko~ide, for example sodium methoxide~
' or an alkali metal hydride such as sodium hydride, me condensation of
the compounds of formulae III and IV yields a compound of the formula,
fOOC~H5
~MHCH2CH20CH2CH2~ - NHR3 V
~OOC H '~'
wherein ~ and R3 are as described above and can be the
same or different protecting groups.
Suitable nitrogen protecting groups for the purposes of the
above discussed condensation reaction include acyl groups such as acetyl,
sulfonyl groups such as tosyl or mesyl, the carbobenzoxy group or the
phthali~ido group. It is understood that if ~ and R3 signifies a
phthalimido group, the nitrogen atom does not carry a hydrogen atom.
me condensation of the compounds of formulae III and IV can be
conducted without a sol~ent system or in the presence of,an inert organic
solvent. Suitable solvents for this purpose include alcohols such as
methanol, ethanol and the like,e~ s such as tetrahydrofuran, and
dimethylformamide (DMF, with DMF being the preferred solvent. If R2
signifies chlorine, it may be expedient to add potassium iodide to the
reaction mixture so that the more reactive iodide ion replaces the chloride
ion. ~his reaction is effected at elevated temp,eratures ,~i,t,h a temperature
', in the range of from about 120 to about,l60 C being prefer,red.,
, The protecting groups, present in ,the resulting comp,ou,nd of
.. ... .
, formula V are then removed to yield D,L,2-amino-4-(2-amino-ethoxy)-butanoic
-5-
~. .. . . .
~ 4~2460
acid. Removal Of the protecting groups i3 accomplished following
conventional techniqucs For example~ the compound of formula V wherein
the protecting groups are phthalimido groups can be treated with an aqueous
mineral acid, such as hydrochloric acid, to effect acid hydrolysis of the
phthalimido groups. If the protecting groups present in the formula V
compound are carbobenzoxy groups, the N-protective group can be removed
either by hydrogenolysis or by treatment with hydrogen brom~de in acetic
acid. If the N-protective group is a tosyl group, it can be removed~
for example, by reductive cleavage with sodium in liquid ammonia.
In a still further process aspect resolution of the racemic
compound, e.g~ obtainad as described above, is accomplished by first
preparing the racemic diacylated product. Thus, D~L,2-amino-4_
(2-aminoethoxy)-butanoic acid is treated with a conventional acylating
agent, such as ac~tic anhydride, acetyl chloride, chloroacetyl chloride,
and the like. me diacylated product thus obtained is then incubated with
a suitable acylase, such as hog renal acylase, which resolves the racemic
compound into a mixture of D and L compounds. The L-compound of formula
I above and the corresponding D-antipode can then be obtained by
separating the D and L materials resulting from the acylase incubation,
and removing the acyl groups by acid hydrolysis using for example aqueous
hydrochlorid acid. In the final crystallization of the L-compound of
formula I, this compound can be obtained as a zwitterion or as its
mono- or di-acid salt, for example as its mono- or di-hydrochloride.
The novel compound of formula I above ~o~msbiologically acceptable
acid addition salts with organic or inorganic acids. Suitable acids for
this purpose include the hydrohalic acids such as hydrochloric acid and
hydrobromic acid, other mineral acids such as sulfuric acid, phosphoric
acid, nitric acid and the like~ and organic acids such as tartaric acid,
citric acid, acetic acid, formic acid, maleic acid, succinic acid and the
like.
--6--
... . . . . .
;~ :
.. : ' `.
~4~4~;0
Thc compound of formula I above either alone or in combination
with ascorbic acid enhan~es ethylene production in fruits and therefore is
useful as a plant growth regulator, especially as an abscission agent.
The role of ethylene in the ripening of fruits has been recognized
in the art for over 30 years. It is known that the production of ethylene
in maturing fruits increases while the fruit separates from its pedicel,
mis knowledge can be utilized to demonstrate the efficacy of an abscission
compound in regard to its influence on fruit to accelerate or increase the
production of ethylene. Since oranges can be considered a typical fruit
representative of those amenable to treatment by chemical abscission
agents, the efficacy of the compound of formula I as an abscission agent
; may be illustrated with respect thereto.
The ability of the compound of formula I above to enhance ethylene
production is demonstrated in the following test procedure. Whole fresh
gree~ oranges and semi-ripe yellow oranges were sprayed with a 0.1% solution
of the compound of formula Io The control oranges were treated with
distilled water. All sprays contained 001% Aerosol~ OT (sodium dioctyl
sulfosuccinate, SDS) as the wetting agent. SDS in the solutions assured
in a thin ~ilm like coverage on the waxy skin of the oranges thereby
producing an increased surface area for the absorption of the active mate-
rial. me treated fruits were then placed in polyethylene bags, glass
jars or aluminum cans and sealed. Air space of the bags or cans was
sampled periodically for ethylene with gas tight syringes.
Ethylene production in the reaction vessels was determined
by gas chromatography. A Hewlett Packard, Series 5750B~ Dual Fla~e
Detector Research Gas Chromatograph was ~itted with lOt x 1/8" stainless
~` steel column packed with alumina (5~ H20 on neutral alumina) to determine
ethylene. The instrument was operated at 45 C. The carrier gas was
helium with a flow rate of 25 ml per minuteO
.
The ethylene nature of the gas measure was established by
--7--
~TRADEMARK
~',-. . ~ . .
. f: .
46(;~
r~tention time in rela~ion to pure ~thylene and it was confirmed by
mercuric perchlorate [Hg~C104)~ reaction and mass spectrometry. Mercuric
perchlorate absorb~ ethylene while hydrochloric acid (HCl~ raleases the
absorbed gas. I'he disappearance and the reappearance of the ethylene
component of the gas was noted in sampl~s treat~,d first with 1-2 ml
0.2M Hg(C104)2 and then with 1 ml 2N HCl.
The mass spectrum of pure ethylene gas was found to be comparable
to the component measured as such andthe ethylene nature of the gas
produced in the reaction vessels was established.
The results for this test procedure for the green oranges are
reported in Table 1. These results show that in the control sample a
slight amount of ethylene was produced while the rate of éthylene
production was greatly enhanced in the oranges treated with the compound
of formula I. me amount of ethylene produced is expressed in microliters
of e~hylene/ml.
Table 1
Ethylene production by green oranges sprayed 1-1.3 gms/
orange incubated in polyethylene bags at room-temperature for 14 days.
Sam~ /ul E/ml
Orange + ~istilled water 000008
Orange ~ 0 1% L-2-amino-4-
(Z-aminoethoxy)-butanoic acid 0.0050 ;~
The results for this test proc~dure for the semi-ripe yellow
oranges are reported in Table 2. These results shown in Table 2 are similar
to those in Table 1 in that again the compound of formula I greatl~
enhanced the rate of ethylene production in the oranges.
-8-
. . . .
.:.. :
... . .
~l~4~ iO
Table 2
.
Ethylene production by yellow oranges sprayed 1-1~3 gms/
or,ange incubated in metal cans at room-tem~erature _ _
~ul E/ml
8 da~s
Orange + Distilled water 0.0009 0.0007
Orange ~ 0,1% L-2_amino_4-
(2_aminoethoxy)-butanoic acid 0.0015 000044
As indicated by the above test procedures~ the compound of
formula I above is useful as a ripening agent or as an abscission agent in
fruit. The results discussed above show that the compound of formula I
when administered as the sole abscission agent enhances ethylene production.
In addition, it has been found that a synergistic effect is obtained from
' combinati~ns of the compound of formula I and ascorbic acid for abscission
of fruit. This synergistic effect is demonstrated by repeating the same ,~
,- test procedures described above using green oranges~ spraying the oranges
^` 10 with
Treatment l-distilled water
Treatment 2-0.1% L,2-amino-4-(2-aminoethoxy)-butanoic acid
,
Treatment 3-Formulation A (ascorbic acid-58.9%~ Disodium-
phosphate 4.1%~,monosodium phosphate-33.9%~ copper sul~ate -
0.1% and aerosol OT-3.0~percentages in weight by weight) at
4% ascorbic acid
Treatment 4-Formulation A at 4% ascorbic a~id and 0.1% L,2-
amino_4_(2~am,inoethoxy)~butano,ic acid. ,
, , The result for this test procedure is set forth in Table 3 be-
' 20 low. From these results, it can be,seen ~that a synergis,tic effect is ob-
tained from the,combination of the compound of formula I and ascorbic acid
for abscission of Pruit.
_g_
2~60
Table 3
Ethylene production by green oranges sprayed 1-1.3 gms/
Sample
Treatment 1 0.0008
Treatment 2 0.0050
Treatment 3 0.0032
Treatment 4 0.0225
The synergistic effect between the compound of formula I above
and ascorbic acid for abscission of citrus is also demonstrated in the
following field trials. These trials were conducted at Vero Beach,
Florida and the applications of the test materials were made in early
May. Selected branches of ~alencia orange trees were sprayed~ each treat-
ment was replicated twice using branches on different trees. Treatments
were applied to run-off using a one quart carbon dioxide sprayer equipped
with a single 800~ Tee Jet nozzle and a pressure of 2,3 atm. The non-
toxic surfactant X-77 spreader (Colloidol Products Corp., Saulsalito,
California) was added to all spray solutions at 0.5%~ me composition
of the sprays applied to the branches were as follows:
Treatment 1~0.4%-L,2-amino-4-~2 aminoethoxy)-butanoic acid
Treatment 2-l~O~o ascor~ic acid
Treatment 3-0.1%-L,2-amino-4-(2-aminoethoxy)-butanoic
acid ~ O~l~o ascorbic acid
Treatment 4-0.2%-L,2-amino-4-(2 aminoethoxy)-butanoic
acid ~ 0.2%o ascorbic acid
The only rainfall that occurred during the 14 day treatment
period feel on the ninth day and amounted to 1,96 cmO me ma~imum and
minimum temperatures for the test period were as follows:
-10_
: .: .: .
:
1 7~ ~ 73
; 2 79 75
3 80 72
4 80 73
~ 5 79 73
; 6 77 60
7 80 72
8 83 76
'9 86 7~7~
8S 76
11 86 73
i 12 90 75
13 88 78
14 90 76 .
me number of fruit was counted on each branch at time of
treatment and at 7 and 14 days after treatmentO The data on the amount
of fruit abscission 7 and 14 days after treatment are presented in Table 4.
Table 4
Average percent ~) F _ t abscission of Valencia oran~s
Sample ~ ys
Untreated 0 0
Treatment 1
Treatment 2 11 22
Treatment 3 '0 25
Treatment 4 43 57
(a) average of two replications
4~46~
Composi-tions containing th0 compound of formula I and ascorbic
acid can be applied to the fruit-bearing trees in liquid or powder formu~
lations. Application may be made to the roots, trunks, limbs, leaves
or fruit. For example, the abscission compositions can be dusted on the
trees fro~ airplanes or applied to the base of the trees in order to be
absorbed by the roots. The preferred method of application and ths most
efficient is to apply the compositions to the trees rom abo~e in the form
of an aqueous spray. If desired, an oily spray may be used.
In order to achieve the most efficient use of the abscission
compositions~ it is preferred to apply them from about 2 to 7 days prior
to harvesting of the mature fruit. It is preferred to incorporate a
conventional adhesive agent into the abscission compositions of the
invention as a precaution against a rainfall occurring after application
and washing the abscission composition from the fruit. Examples of
such adhesive agents include glue~ casein, salts of alginic acids~ cellu-
lo~e ~ums and their derivatives, polyvinyl pyrrolidone, vegetable gums~
propylene glycol, invert syrup, corn syrup and the like.
These abscission compositions can contain, in addition to the
compound of formula I and ascorbic acid, a water-soluble cupric salt
such as cupric sulfate, cupric chloride and the like, a buffer and a
surfactant. If desired, inert materials conventionally used in agriculture
for applications to trees may be utilized.
In order to form the liquid spray formulations for the abscission
compositions the active ingredients are dispersed in a carrier such as,
for example, water or other suitable liquids. In liquid spray compositions,
it is preferred to include from about 0.1% to about 0.5% by weight, based
on the weight of the carrier, of a surface active agent The surface
active agents may be anionic, cationic or non-ionic in character~ ~pical
classes of surface active agents include alk3~lsulfonates, alklarylsulfonates,
alk~lsulfates, alkylamidesulfonates~ alkylaryl polyether alcohols, fatty
-12_
. ' ' ' '.' . " . .` .. ': `' ' . '.
~4;~6~)
acid esters of polyhydric alcohols, ethylene oxide addition products of
such esters; addition products of long chain mercaptans and ethylene
oxide; sodium alkyl benæene sulfonates having from 14 to 18 carbon atoms,
alkylphenylethylene oxides, e.g. paranonylphenol condensed with lO ethylene
oxide l~ts or parais~octyl phenol condensed with 10 ethylene oxide units
or with two ethylene oxide units or with 16 ethylene oxide units, and soaps,
e.g. sodium stearate and sodium maleate~ Typical surface active agents are:
sodium salt of propylated naphthalenesulfonic acids; Aeroso~ OT manufactured
by American Cyanamide Co. New York, New ~ork; X-77 Spreader manufactured by
Colloidal Products Corp~, Saulsalito, California; the sodium salt of modified
alcohol sulfate from coconut fatty acids; the sodium salt of sulfonated
monoglyceride of coconut fatty acids; the sodium sulfonate of butylbisphenyl
sorbitan sesquiolate; lauryltrimethyl ammonium chloride; octadecyltrimethyl
ammonium chloride; polyethyleneglycol laurylether; Daxad~ No. 11 manufactured
by Dewey and Almy Chemical Co., Cambridge, Mass. (sodium salt of polymerized
aIkyl aryl sulfonic acid3; sodium oleate sulfate; sodium lauryl sulfateg
Ethofats~ manufactured by Ar~our ~ Co. Chicago, ILl~ ~polyethylene esters ~;
of fatty acids or rosin acids); Ethomeens~ manufactured by Armour ~ Co.,
Chicago~,Ill. (polyethylene glycol derivatives of long chain alkylamines)g
20 Tritons* manufacture~d by Rohm ~ Hass Co., Philadelphia, Pa. (alkylaryl ~-
polyether alcohols, sulfonates, and sulfates of the non-ionic, cationic and
anionic types) and the likeO
These abscission compositions can be used to abscind a variety of
fruits from trees. Typical fruits with which these compositions are
efficacious include oranges, olives, apples, cherries and the like. The
compositions of the invention are most efficacious in the abscission of
citrus fruits, e.g., oranges, grapefruit and the like.
If, under particular application conditions, it is desirable to
adjust the pH of the abscission compositions, th~s can be done following
conventional techniques. For example, buffers such as disodium phosphate,
*TRADEMARK
-13
: : :
~04~9~6~
monosodi~ phosphate, sodium dibasic phosphate monohydrate and the like
or mixtures of these can be incorpo~ated into -the abscission compositions
to adjust the pH to the desired range.
The nature and objects of the present invention can be more
fully understood by making reference to the following examples~ Unless
otherwise indicated, all temperatures are given in degrees Centigrade.
Example 1
Prepa,ration of LvZ-a~ino-4-~2-a noe,tho ~ u,tan,,oic acid
10 ~a~
A solution of 125 mg of L,trans-2-amino-4-(2-aminoethoxy)-3-
butenoic acid in S0 ml 90% methanol-water was reduced with H2 at 1
atmosphere and 25 in the presence of 150 mg 5% Pd on charcoal~ me
catalyst was removed by filtration, the filtrate partially evaporated
under reduced pressure and the concentrate applied to 5 ml Biorad~'~ AG50X4
(100-200 mesh) cation exchange resin in the H+ form. The resin was then
eluted successivel~ with 10% pyridine solution and 1 M NH40H solution.
The eluates were evaporated to dryness under reduced pressure.
Thin layer chromatography of the pyridine e-luate indicated that
the major ninhydrin positive component was 2-aminobutanoic acid. The
ammonia eluate residue was taken up in a small volume of H20~ the pH was
adjusted to 3.8 with 1 N HCl the residue concentrated to dryness and the
above-identified product was crystallized from 2 ml methanol, ~pO 205-207 .
Example 2
,` ~_=
~ydrochloride.
A solution of 125 mg. of L-trans-2-amino-4-(2-aminoethox~t3
butenoic acid in 50 ml 90% methanol-water was reduced with H2 at 1
atmosphere and 25 in the presence of 75 mg 10% platinum on charcoal.
*TRADEMARK -14-
,
4~iO
The catalyst was removed by filtration, the filtrate partially evaporated
under reduced pressure and the concentrate applied to 5 ml. Biora~
A~SOX4 ~100-200 mesh~ cation exchange resin in the H~ form. The resin
was then eluted successively with 10% pyridine solution and lM NH40H
solution. The eluates were evaporated to dryness under reduced pressure.
Thin layer chromatography of ~he pyridine eluate indicated that the major
ninhyd~in positive component was 2-aminobutanoic acid. me ammonia eluate
residue were taken up in a small volume of water, the pH was adjusted to
3.8 with lN HCl, the residue concentrated to dryness and the above_
identified product was crystalli~ed from 2 ml of methanolJ m.p. 205-207 .
Example 3
Preparation of L-2-a } ~ oethoxy)-butan~oic acid
h~drochloride.
A solution of 7.85 g of L,trans-2-amino-4-(2-aminoethoxy)-3-
butenoic acid (40 mmoles) in 200 ml H20 was treated in a Parr apparatus
at 1 atmosphere pressure and 25 for 2 hours with hydrogen in the presence
of 1 g 5% Pd on charcoal. The solution was then filtered and the product
was adsorbed on 70 ml AG50WX-4 cation exchange resin (100-200 mesh in
the H~ form~3 The resin was eluted with 10% aqueous pyridine solution and
the eluate was evaporated under reduced pressure to a 24 mg residue. The
resin was then eluted with lM NH~OH, the eluate partially evaporated under
reduced pressure, the concentrate adjusted to pH 5.0 with 18 ml 2N HCl
and the remaining solvent evaporated under reduced pressure. The residue
was taken up in hot methanol and the above-identified product
crystallized after addition of ethanol m.p. 208-211 .
;
~TRADE~RK
-15-
~0~;~4~
Example 4
Preparation of D,L-2 amino-4-(2-aminoethoxy~-bltanoic
.,~9~
2-Chloroethyl-2~-phthalimidoethyl ether, (37,5 mmole, 9.4 g),
sodium diethyl phthalimidomalonate, (25 ~ole, 8.2 g) and potassium iodide
(2.5 mmoles, 0.4 g) were dissolved in 10 ml dimethylforlllamide and the
solution was maintained at 153 for 4 hours by which time titration of a
small portion indicated that 99% of the malonate reagent had reacted. The
product, diethyl 2-phthalimido-2-(phthalimidoethoxyethyl)-malonate could
be crystallized from ethanol, m.pO 96-98 , However it was more efficient
to proceed by precipitating the c~de condensation product by addition oP
4 volumes of water and triturating the precipitate 2 times with 40 ml
waterO
The crude condensation product from 4/5 of the original reaction
mixture was dissolved in 20 ml ethanol, 40 ml of an aqueous solution of
5 N NaOH was added and the solution was refluxed for 1 hour. The ethanol
was then allowed to boil off and the cooled solution was adjusted to pH l
with 6 N HCl. The aqueous phase was decanted from the oil which formed, and
the oilw~s~r~luxed in 120 ml 6 N HCl for 90 minutes. After cooling~-and
filtering~ the solvent was removed by evaporation under reduced pressure
and the residue was dissolved in water and appl;ed to 100 ml Bio-Rad
AG50X4 (50-100 mesh) cation exchange resin in the ~rform. The resin was
eluted first with 100 ml 20% aqueous pyridine solution and then with 200 ml
aqueous 1 N NH40H. The latter eluate was partially evaporated under reduced
pressure and the pH was then adjusted to 305 with 22 meq HCl. The water was
removed by evaporation under reduced pressure, the residue dissolved!,iin a
small amount of methanol, and after addition of ethanol to a total volume
of 50 ml7 the above-identified product crystallized during storage at
0~ miPo 175-177o
l.
-16-
:`
:~V~6~)
Example 5
Resolution ~ ~
A solution of D,L-2-amino-4-(2-aminoethoxy)butanoic acid, lg
(5 mmole) in 5 ~l 2 N NaOH, was treate~ with 1.25 ml, 11 mmoles~
chloroac0tylchloride and the pH was maintained at 10.2 by the ad~ition
of 2 N NaOH while the temperature was kept at 5 . After 45 minutes the
pH was adjusted to 1.0 with 2 N ~Clo The product did not precipitate but
was extracted with 5 X 10 ml ethyl ether. The combined extracts were back
extracted with lO ml H20 and the organic phase evaporated at reduced pres-
; 10 sure to 1.6 g syrup. The syrup was taken up in ethanol and a saturated
solution of LiOH was added to an apparent pH of 7. The solvent was
evaporated under reduced pressure to yield the lithi~ salt of D,L-2-
chloroacetylamino-4-(2~chloroacetyl-aminoethoxy)-butanoic acid, which was
crystallized from 5 ml 50% ether-ethanol, m.p. 201-203 .
solution of 642 mg of the so-obtained di-acylated product in
20 ml deionized water was treat~d with 30 mg hog renal acylase at 37 and
pH 7.2 for 21 hours. The solution was then applied to a column containing
10 ml Biorad AG5~WX4 cation exchange resin l50-loo mesh in the H+ form~. -
The colu~n effluent plus a 50 ml water wash was concentrated to 20 ml,
the pH readjusted to 7.2 with LiOH solution, and additional 30 mg acylase
added and another incubation carried out for 8 hours. The solution was
then reapplied to the same column and the column effluent and watar wash
were again put through the same procedure. From the final column effluent
and wash, the lithium salt was made and ~-2-chloroacetylamino-4 (2-chloro-
acetylaminoethoxy)butanoic acid crystallized from ethanol after removing
the acylase by filtration from an aqueous ethanol suspension. After
recrystallization the product showed a m.p. of 210 .
The above described resin was then eluted with 100 ml 10%
aqueous pyridine solution. The eluate was concentrated under reduced
pressure and L_2-amino 4-~2-chloroacetylaminoethoxy)-butanoic acid was
-17-
.: . - . . . : , :
.. . . .
~04~46iO
crystallized from ethanol water, m.p~ after recrystallization 139-141 .
The chloroacetyl groups were removed from 'both D-2~chloro-
acetylamino-4-(chloroacetylaminoethoxy)butanoic acid and L_2-amino-4-
~2-chloroacetylam1noethoxy)butanoic acid by refluxing 0.~ mmole of each
for 2 hours in 10 ml 2N HCl~ After evaporation at reduced pressure ea'ch
preparation was taken up in water and applied to a 5 ml column of Biorad
AG50WX4 (50-100 mesh) cation exchange resin in the H f'~orm. After washing
the columns with aqueous pyridine solution, the products were eluted with
50 ml 1 N NH40~. The solvent was partially evaporated under reduced pres-
sure, the pH adjusted to 4.5 with 1 N HCl and the products crystalli~.edfrom ethanol-water. Thus, from D-2~chloroacetylamino-4-(2-chloroacetyl-
aminoethoxy)-butanoic acid there was obtained D-2-amino-4-(2-aminoethox~)
butanoic acid hydrochloride, m.p. 206 and from L_2-amino-4-(2-chloro-
acetylaminoethoxy)-butanoic acid there was obtained L,2-amino-4-(2-
aminoethoxy)-butanoic acid hydrochloride, m,p, 204-Z06 .
~.`
. .
-18-
': ' . . . . . . ' ' ' ., ' ': ~:
