U.S. patent application number 14/691813 was filed with the patent office on 2015-08-13 for preparation and use of methionylmethionine as feed additive for fish and crustaceans.
This patent application is currently assigned to EVONIK DEGUSSA GmbH. The applicant listed for this patent is Thomas HAEUSSNER, Christoph KOBLER, Christoph WECKBECKER. Invention is credited to Thomas HAEUSSNER, Christoph KOBLER, Christoph WECKBECKER.
Application Number | 20150223495 14/691813 |
Document ID | / |
Family ID | 41396092 |
Filed Date | 2015-08-13 |
United States Patent
Application |
20150223495 |
Kind Code |
A1 |
KOBLER; Christoph ; et
al. |
August 13, 2015 |
PREPARATION AND USE OF METHIONYLMETHIONINE AS FEED ADDITIVE FOR
FISH AND CRUSTACEANS
Abstract
An animal feed mixture containing DL-methionyl-DL-methionine and
salts thereof for animals kept in aquacultures is provided. Methods
for preparing DL-methionyl-DL-methionine of formula (I)
##STR00001## and methods to fractionate the diasteriomeric forms
obtained are also provided.
Inventors: |
KOBLER; Christoph; (Alzenau,
DE) ; HAEUSSNER; Thomas; (Eppertshausen, DE) ;
WECKBECKER; Christoph; (Gruendau-Lieblos, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
KOBLER; Christoph
HAEUSSNER; Thomas
WECKBECKER; Christoph |
Alzenau
Eppertshausen
Gruendau-Lieblos |
|
DE
DE
DE |
|
|
Assignee: |
EVONIK DEGUSSA GmbH
Essen
DE
|
Family ID: |
41396092 |
Appl. No.: |
14/691813 |
Filed: |
April 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13616533 |
Sep 14, 2012 |
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14691813 |
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12580283 |
Oct 16, 2009 |
8968817 |
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13616533 |
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61117361 |
Nov 24, 2008 |
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Current U.S.
Class: |
426/2 |
Current CPC
Class: |
C07C 319/20 20130101;
C07C 319/20 20130101; A23K 50/80 20160501; C07D 241/08 20130101;
Y02P 20/582 20151101; Y02A 40/818 20180101; A23K 20/142 20160501;
C07C 323/60 20130101 |
International
Class: |
A23K 1/16 20060101
A23K001/16; A23K 1/18 20060101 A23K001/18 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 17, 2008 |
DE |
102008042932.5 |
Claims
1-9. (canceled)
10. A method for feeding an animal, comprising feeding the animal a
feed mixture comprising a nutrient selected from the group
consisting of DL-methionyl-DL-methionine, a salt thereof and a
mixture of DL-methionyl-DL-methionine and a salt thereof, wherein
the animal is kept in an aquaculture.
11. The method as claimed in claim 10, where the animal kept in an
aquaculture is at least one animal selected from the group
consisting of a salt water fish, a salt water crustacean, a fresh
water fish and a fresh water crustacean.
12. The method as claimed in claim 10, wherein the animal kept in
an aquaculture is an animal selected from the group consisting of a
carp, a trout, a salmon, a catfish, a perch, a flatfish, a
sturgeon, a tuna, an eel, a bream, a cod, a shrimp, a hill and a
prawn.
13. The method as claimed in claim 10, wherein the animal is
selected from the group of animals consisting of a silver carp
(Hypophthalmichthys molitrix), a grass carp (Ctenopharyngodon
idella), a common carp (Cyprinus carpio), a bighead carp
(Aristichthys nobilis), a carassius (Carassius carassius), a catla
(Catla Catla), a Roho labeo (Labeo rohita), a Pacific salmon
(Salmon salar) an Atlantic salmon (Oncorhynchus kisutch), a rainbow
trout (Oncorhynchus mykiss), an American catfish (Ictalurus
punctatus), an African catfish (Clarias gariepinus), a pangasius
(Pangasius bocourti and Pangasius hypothalamus), a Nile tilapia
(Oreochromis niloticus), a milkfish (Chanos chanos), a cobia
(Rachycentron canadum), a whiteleg shrimp (Litopenaeus vannamei), a
black tiger shrimp (Penaeus monodon) and a giant river prawn
(Macrobrachium rosenbergii).
14. The method according to claim 10, wherein the nutrient is a
salt of DL-methionyl-DL-methionine and the salt comprises a cation
selected from the group consisting of an alkali metal, an alkaline
earth metal, ammonium, Cu.sup.2+, Zn.sup.2+, Co.sup.2+ and a
mixture thereof.
15. The method according to claim 10, wherein a content of the
nutrient in the animal feed mixture is from 0.01 to 5% by
weight.
16. The method according to claim 10, wherein the feed mixture
further comprises: protein and carbohydrate, obtained from a meal
selected from the group consisting of fish meal, soybean meal, corn
meal, and mixtures thereof, and, optionally, a supplement selected
from the group consisting of an essential amino acid, a protein, a
peptide, a vitamin, a mineral, a carbohydrate, a fat, an oil and a
mixture thereof.
17. The method according to claim 10, wherein the
DL-methionyl-DL-methionine or salt thereof comprises one mixture
selected from the group consisting of a DD/LL/LD/DL mixture, a
DL/LD mixture, and a DD/LL mixture.
18. The method according to claim 10, wherein the feed mixture
further comprises 0.01 to 20% by weight DL-methionine.
19. The method according to claim 10, wherein the
DL-methionyl-DL-methionine is a DL/LD-methionylmethionine pair of
enantiomers.
20. The method according to claim 22, wherein the feed mixture
further comprises 0.01 to 20% by weight DL-methionine.
21. The method according to claim 10, wherein the feed mixture is
in the form of a pellet or an extrudate.
22. The method according to claim 10, wherein the nutrient is
solely a DL/LD methionylmethionine pair of enantiomers.
23. The method according to claim 10, wherein the nutrient does not
comprise a DD enantiomer of DL-methionyl-DL-methionine or a salt
thereof
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is continuation of U.S. application Ser.
No. 13/616,533 filed Sep. 14, 2012, pending, which is a divisional
of U.S. 12/580,283 filed Oct. 16, 2009, U.S. Pat. No. 8,968,817,
and claims the benefit of U.S. 61/117,361 filed Nov. 24, 2008 and
DE 10 2008 042 932.5 filed Oct. 17, 2008.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to novel chemical syntheses of
methionylmethionine, the dipeptide of methionine, and the specific
use thereof as feed additive alone or mixed with methionine for
fish and crustacean nutrition.
[0004] 2. Description of the Related Art
[0005] Essential amino acids (EAA) such as methionine, lysine or
threonine are very important constituents as feed additives in
animal nutrition and play a significant part in the commercial
rearing of productive animals such as, for example, chickens, pigs
and ruminants. Supplementation of natural protein sources such as,
for example, soybeans, corn and wheat with EAAs makes it possible
on the one hand for the animals to grow faster, or for milk
production to be higher in high-output dairy cows, but on the other
hand for the utilization of the feed to be more efficient. This
represents a very great commercial advantage. The markets for feed
additives are of great industrial and commercial importance. In
addition, they are high-growth markets, attributable not least to
the increasing importance of countries such as, for example, China
and India.
[0006] L-Methionine ((S)-2-amino-4-methylthiobutyric acid)
represents the first limiting amino acid for many species such as
chickens, ducks, turkeys and also for many fish and shellfish
species and therefore plays a very significant part in animal
nutrition and as feed additive (Rosenberg et al., J. Agr. Food
Chem. 1957, 5, 694-700 and Lovell, T. R., J. Anim. Sci. 1991, 69,
4193-4200). However, in the classical chemical synthesis,
methionine results as racemate, a 50:50 mixture of D- and
L-methionine. This racemic DL-methionine can, however, be employed
directly as feed additive because there is in some species under in
vivo conditions a transformation mechanism which converts the
unnatural D enantiomer of methionine into the natural L enantiomer.
This entails firstly the D-methionine being deaminated with the aid
of a nonspecific D-oxidase to .alpha.-ketomethionine, and
subsequently being further transformed with an L-transaminase into
L-methionine (Baker, D. H. in "Amino acids in farm animal
nutrition", D'Mello, J. P. F. (ed.), Wallingford (UK), CAB
International, 1994, 37-61). The available amount of L-methionine
in the body is increased thereby and can then be available to the
animal for growth. The enzymatic transformation of D- to
L-methionine has been detected in chickens, pigs and cows, but
especially also in carnivorous and omnivorous fish and also in
shrimps and prawns. Thus, for example, Sveier et al. (Aquacult.
Nutr. 2001, 7 (3), 169-181) and Kim et al. (Aquaculture 1992, 101
(1-2), 95-103) were able to show that the transformation of D- into
L-methionine is possible in carnivorous Atlantic salmon and rainbow
trout. Robinson et al. (J. Nutr. 1978, 108 (12), 1932-1936) and
Schwarz et al. (Aquaculture 1998, 161, 121-129) were able to show
the same for omnivorous fish species such as, for example, catfish
and carp. In addition, Forster and Dominy (J. World Aquacult. Soc.
2006, 37 (4), 474-480) were able to show in feeding experiments on
omnivorous shrimps of the species Litopenaeus vannamei that
DL-methionine has the same activity as L-methionine.
[0007] The world production in 2007 of crystalline DL-methionine
and racemic, liquid methionine hydroxy analog (MHA,
rac-2-hydroxy-4-(methylthio)butanoic acid (HMB)) and solid calcium
MHA was more than 700,000 t, which was successfully employed
directly as feed additive for monogastric animals such as, for
example, poultry and pigs. Owing to the rapid commercial
development of fish and crustacean farming in highly industrialized
aquacultures an optimal, economical and efficient methionine
supplementation option has become increasingly important precisely
in this area in recent years (Food and Agriculture Organization of
the United Nation (FAO) Fisheries Department "State of World
Aquaculture 2006", 2006, Rome, International Food Policy Research
Institute (IFPRI) "Fish 2020: Supply and Demand in Changing
Markets", 2003, Washington, D.C.). However, in contrast to chickens
and pigs, various problems occur on use of methionine, MHA or
Ca-MHA as feed additive for certain fish and crustacean varieties.
Thus, Rumsey and Ketola (J. Fish. Res. Bd. Can. 1975, 32, 422-426)
report that the use of soybean meal in conjunction with singly
supplemented crystalline amino acids did not lead to any increase
in growth of rainbow trout. Murai et al. (Bull. Japan. Soc. Sci.
Fish. 1984, 50, (11), 1957) were able to show that daily feeding of
fish diets with high rates of supplemented crystalline amino acids
in carp led to more than 40% of the free amino acids being excreted
via the gills and kidneys. Because of the rapid absorption of
supplemented amino acids shortly after feed intake, there is a very
rapid rise in the amino acid concentration in the fish's blood
plasma (fast response). However, at this time, the other amino
acids from the natural protein sources such as, for example,
soybean meal are not yet present in the plasma, possibly leading to
asynchronicity of the concurrent availability of all the important
amino acids. As a result thereof, part of the highly concentrated
amino acids is rapidly excreted or rapidly metabolized in the body,
and is used for example as pure energy source. As a result, there
is only a slight or no increase in growth, upon use of crystalline
amino acids as feed additives (Aoe et al., Bull. Jap. Carp Soc.
Sci. Fish. 1970, 36, 407-413). Supplementation of crystalline amino
acids may lead to further problems in crustaceans. The slow feeding
behavior of certain crustaceans such as, for example, shrimps of
the species Litopenaeus Vannamei results, owing to the long
residence time of the feed under water, in the supplemented,
water-soluble amino acids being dissolved out (leaching), leading
to eutrophication of the water and not to an increase in growth of
the animals (Alam et al., Aquaculture 2005, 248, 13-16).
[0008] Efficiently supplying fish and crustaceans kept in
aquacultures thus requires, for certain species and applications, a
specific methionine product form, such as, for example, an
appropriately chemically or physically protected methionine. The
aim of this is on the one hand that the product remains
sufficiently stable in the aqueous environment during feeding and
is not dissolved out of the feed. On the other hand that the
methionine product eventually taken in by the animal can be
utilized optimally and with high efficiency in the animal body.
[0009] Many efforts have been made in the past to develop suitable
feed additives, particularly based on methionine, for fish and
crustaceans. Thus, for example, WO8906497 describes the use of di-
and tripeptides as feed additive for fish and crustaceans. The
intention of this is to promote the growth of the animals. However,
the di- and tripeptides preferably employed in this case were from
nonessential and therefore also nonlimiting amino acids such as,
for example, glycine, alanine and serine. The only
methionine-containing dipeptides described are
DL-alanyl-DL-methionine and DL-methionyl-DL-glycine. However, this
means that effectively only 50% of active substance (mol/mol) are
present in the dipeptide, and this must be categorized as very
disadvantageous from the aspect of economics. WO02088667 describes
the enantioselective synthesis and use of oligomers of MHA and
amino acids such as, for example, methionine as feed additives,
inter alia also for fish and crustaceans. It is said to be possible
to achieve faster growth thereby. The described oligomers are
assembled by an enzyme-catalyzed reaction and exhibit a very broad
distribution of the chain lengths of the individual oligomers. This
makes the process unselective, costly and elaborate in the
procedure and purification. Dabrowski et al. describes in
US20030099689 the use of synthetic peptides as feed additives for
promoting the growth of aquatic animals. In this case, the
proportion of the peptides in the complete feed formulation may be
6-50% by weight. The synthetic peptides preferably consist of
essential and limiting amino acids. However, the synthesis of such
synthesized oligo- and polypeptides is very elaborate, costly and
difficult to convert to the industrial scale. In addition, the
effectiveness of polypeptides of a single amino acid is disputed,
because these are often converted only very slowly or not at all
under physiological conditions into free amino acids. Thus, for
example, Baker et al. (J. Nutr. 1982, 112, 1130-1132) describes the
lack of biological value of poly-L-methionine in chickens because
of the absolute insolubility in water, since absorption by the body
is impossible.
[0010] Besides the use of novel chemical methionine derivatives
such as, for example, methionine-containing peptides and oligomers,
there has also been investigation of various physical protection
possibilities such as, for example, coatings and the incorporation
of an amino acid in a protective matrix. Thus, for example, Alam et
al. (Aquacult. Nutr. 2004, 10, 309-316 and Aquaculture 2005, 248,
13-19) were able to show that coated methionine and lysine has, in
contrast to uncoated, a very positive influence on the growth of
young kuruma shrimps. Although use of a specific coating was able
to suppress the leaching of methionine and lysine out of the feed
pellet, there are some serious disadvantages. The preparation or
the coating of methionine usually represents a technically
complicated and elaborate process and is therefore costly. In
addition, the surface coating of the methionine after coating is
easily damaged by mechanical stress and abrasion during feed
processing, possibly leading to a diminution or complete loss of
the physical protection. An additional factor is that the content
of methionine is reduced, and thus often becomes uneconomic, by a
coating or use of a matrix substance.
[0011] Besides the inventive novel use of
DL-methionyl-DL-methionine as feed additive with low leaching
characteristics from feed pellets and extrudates, and an optimal
supply of methionine to the body through slow-release cleavage of
methionylmethionine, it has also been possible to develop novel
processes for preparing methionylmethionine which have many
advantages over the preparation variants described in the
literature. Most of the dipeptide syntheses disclosed in the
literature use costly protective groups such as, for example,
Boc-(tert-butoxycarbonyl) or Z-(benzyloxycarbonyl) protective
groups, which have to be attached to the appropriate amino acid
before the actual dipeptide synthesis, and subsequently eliminated
again. In addition, activation of the amino acids to be coupled is
usually necessary. Thus, methionylmethionine can be prepared by
coupling N-Boc-methionine with the methyl ester of methionine using
dicyclohexylcarbodiimide (DCC). The great disadvantages of this
preparation process are the use of costly protective groups, a very
elaborate synthesis and costly coupling reagents which cannot be
recycled, such as, for example, DCC. Another alternative for the
industrial synthesis of methionylmethionine is described in
DE2261926. 3,6-Bis[2-methylthio)ethyl]-2,5-piperazinedione
(methioninediketopiperazine, DKP) is formed in the first stage by
heating the isopropyl ester of methionine and is then hydrolyzed to
methionylmethionine. Merely satisfactory yields of 62-65% were
possible for the hydrolysis step in this case. In addition, the use
of methionine isopropyl ester as starting material is too costly
and therefore uneconomic.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows a diagrammatic representation of the enzymatic
cleavage of the methionylmethionine diastereomer mixtures DD/LL-I,
DL/LD-I and DD/LL/DL/LD-I.
[0013] FIG. 2 shows a diagrammatic representation of the enzymatic
cleavage of the four methionylmethionine diastereomers DD-I, LL-I,
DL-I and LD-I with different rates of cleavage.
[0014] FIG. 3 shows a diagrammatic representation of the enzymatic
liberation of methionine (D- and L-Met together) from the four
methionylmethionine diastereomers DD-I, LL-I, DL-I and LD-I.
[0015] FIG. 4 shows the biotransformation of D-methionine to
L-methionine with an enzyme cocktail from common carp.
[0016] FIG. 5 shows the leaching characteristics of
methionylmethionine diastereomer mixtures DD/LL-I, DL/LD-I and
LL/DD/LD/DL-I compared with methionine, MHA and MHA-Ca.
[0017] FIG. 6 shows the in vitro digestion of four different
methionylmethionine diastereomers LL-I, LD-I, DL-I and DD-I with
digestive enzymes of the common carp.
[0018] FIG. 7 shows the in vitro digestion of various
methionylmethionine diastereomer mixtures LL/DD-I, DL/LD-I and
LL/DD/LD/DL-I with digestive enzymes of the common carp.
[0019] FIG. 8 shows the in vitro digestion of four different
methionylmethionine diastereomers LL-I, LD-I, DL-I and DD-I with
digestive enzymes of the rainbow trout.
[0020] FIG. 9 shows the in vitro digestion of the
methionylmethionine diastereomer mixtures LL/DD-I, DL/LD-I and
LL/DD/LD/DL-I with digestive enzymes of the rainbow trout.
[0021] FIG. 10 shows the in vitro digestion of four different
methionylmethionine diastereomers LL-I, LD-I, DL-I and DD-I with
digestive enzymes of the whiteleg shrimps.
[0022] FIG. 11 shows the in vitro digestion of various
methionylmethionine diastereomer mixtures LL/DD-I, DL/LD-I and
LL/DD/LD/DL-I with digestive enzymes of the whiteleg shrimps.
DETAILED DESCRIPTION OF THE INVENTION
[0023] It is an object of the present invention to provide a
feedstuff or a feed additive for animal nutrition based on a novel
methionine substitute which can be employed alone or as mixture
with methionine especially in the sector of industrial fish and
crustacean farming in aquacultures. This and other objects have
been achieved by the present invention, the first embodiment of
which includes an animal feed mixture comprising a nutrient
selected from the group consisting of DL-methionyl-DL-methionine, a
salt thereof and a mixture of DL-methionyl-DL-methionine and a salt
thereof.
[0024] A second object of the present invention is to provide a
simple and cost-effective chemical synthesis of this novel
methionine substitute. This objective has also been achieved by the
present invention, a further embodiment of which includes a process
for preparing DL-methionyl-DL-methionine of formula (I),
comprising:
##STR00002##
[0025] reacting a urea derivative of formula II to obtain
DL-methionyl-DL-methionine;
##STR00003##
[0026] wherein the urea derivative of formula II is one derivative
selected from the group consisting of IIa, IIb, IIc, IId, IIe, IIf
and IIg, and
[0027] R.sup.1 and R.sup.2 in the urea derivatives IIa, IIb, IIc,
IId, IIe, IIf and IIg are defined as follows:
IIa: R.sup.1=COOH, R.sup.2=NHCONH.sub.2
IIb: R.sup.1=CONH.sub.2, R.sup.2=NHCONH.sub.2
IIc: R.sup.1=CONH.sub.2, R.sup.2=NH.sub.2
IId: R.sup.1--R.sup.2=--CONHCONH--
IIe: R.sup.1=CN, R.sup.2=OH
IIf: R.sup.1=CN, R.sup.2=NH.sub.2
IIg: R.sup.1==O, R.sup.2=H.
[0028] In the light of the disadvantages of conventional synthesis
methods, an object of the present invention is to provide a
chemically protected methionine product for various omnivorous,
herbivorous and carnivorous fish and crustacean species which live
in salt or fresh water. It is intended in particular that this
product show low solubility characteristics (leaching) from the
complete feed pellet or extrudate in water and possess a
slow-release mechanism, i.e. a slow and continuous release of free
methionine under physiological conditions. In addition, the novel
methionine product of the present invention may be employed
advantageously as a mixture with DL-methionine.
[0029] In a further embodiment, the present invention provides a
methionine substitute as feedstuff or a feed additive which has
very high biological value and which is easy to handle and store
and has good stability under the usual conditions of compound feed
processing, especially pelleting and extrusion.
[0030] In another embodiment, the present invention provides fish
and crustaceans with a further efficient methionine source, besides
crystalline DL-methionine, which source exhibits if possible the
disadvantages of the known products to only a reduced extent or not
at all.
[0031] In a further embodiment, the present invention provides a
novel, flexible synthesis route for methionylmethionine
(DL-methionyl-DL-methionine) in which the typical precursors and
byproducts from the industrial DL-methionine production process may
be used as starting material. In a still further embodiment, the
present invention provides a process for separating the pairs of
diastereomers DD/LL- and DL/LD-methionylmethionine, so that an
optimal and efficient use of only one pair of diastereomers
(DL/LL-I or DL/LD-I) may be possible for specific applications.
[0032] Within the context of the present invention, all ranges
below include explicitly all subvalues between the upper and lower
limits.
[0033] In a preferred embodiment of the present invention,
DL-methionyl-DL-methionine and salts thereof are provided as a feed
additive in feed mixtures for animals kept in aquacultures. The
feed mixture may comprise from 0.01 to 5% by weight, preferably
comprises 0.02 to 3.0% by weight and most preferably comprises from
0.05 to 0.5% by weight of DL-methionyl-DL-methionine.
[0034] The use of DL-methionyl-DL-methionine is particularly
advantageous in this connection because the compound shows
excellent leaching characteristics because of the low solubility of
the mixture of DD/LL/DL/LD-methionylmethionine and of the pair of
diastereomers DL/LD-methionylmethionine (0.4 g/l).
[0035] The compound further shows good pelleting and extrusion
stability during feed production. DL-Methionyl-DL-methionine is
stable in mixtures with conventional components and feedstuffs such
as, for example, cereals (e.g. corn, wheat, triticale, barley,
millet, inter alia), vegetable or animal protein sources (e.g.
soybeans and oilseed rape and the products of the processing
thereof, legumes (e.g. peas, beans, lupins, etc.), fish meal, inter
alia) and in combination with supplemented essential amino acids,
proteins, peptides, carbohydrates, vitamins, minerals, fats and
oils.
[0036] It is a further advantage of the present invention that, one
mole of water is saved per mole of methionylmethionine compared
with DL-methionine owing to the high active substance content of
methionylmethionine per kg of substance.
[0037] In a preferred embodiment, the feed mixture may comprise
proteins and carbohydrates, preferably based on fish meal, soybean
meal or corn meal, and may be supplemented with essential amino
acids, proteins, peptides, vitamins, minerals, carbohydrates, fats
and oils.
[0038] It is particularly preferred for the
DL-methionyl-DL-methionine to be present in the feed mixture solely
as DD/LL/LD/DL mixture, as DL/LD or DD/LL mixture, preferably in
each case additionally mixed with DL-methionine, preferably with a
DL-methionine content of from 0.01 to 20% by weight, most
preferably of from 0.5 to 15% by weight and particularly preferably
of from 1 to 10% by weight.
[0039] In a particularly preferred embodiment of the present
invention, DL-methionyl-DL-methionine may be a
DL/LD-methionylmethionine pair of enantiomers.
[0040] In a preferred method of feeding animals according to the
present invention, the animals kept in aquacultures are fresh and
salt water fish and crustaceans selected from the group consisting
of carp, trout, salmon, catfish, perch, flatfish, sturgeon, tuna,
eels, bream, cod, shrimps, krill and prawns, very preferably for
silver carp (Hypophthalmichthys molitrix), grass carp
(Ctenopharyngodon idella), common carp (Cyprinus carpio) and
bighead carp (Aristichthys nobilis), carassius (Carassius
carassius), catla (Catla Catla), Roho labeo (Labeo rohita), Pacific
and Atlantic salmon (Salmon salar and Oncorhynchus kisutch),
rainbow trout (Oncorhynchus mykiss), American catfish (Ictalurus
punctatus), African catfish (Clarias gariepinus), pangasius
(Pangasius bocourti and Pangasius hypothalamus), Nile tilapia
(Oreochromis niloticus), milkfish (Chanos), cobia (Rachycentron
canadum), whiteleg shrimp (Litopenaeus vannamei), black tiger
shrimp (Penaeus monodon) and giant river prawn (Macrobrachium
rosenbergii).
[0041] According to the invention, DL-methionyl-DL-methionine (I)
(methionylmethionine or Met-Met for short) or its alkali metal and
alkaline earth metal salts such as, for example, the slightly
soluble calcium or zinc salt may be used as addition in feed
mixtures as DD/LL/DL/LD, DD/LL or DL/LD diastereomer mixture, alone
or mixed with DL-methionine, preferably for fish and
crustaceans:
##STR00004##
Four different stereoisomers (diastereomers) exist of the dipeptide
DL-methionyl-DL-methionine (I), DD-, LL, DL- and LD-I, of which
only L-methionyl-L-methionine (LL-I) is natural, all the other
three dipeptides L-methionyl-D-methionine (LD-I),
D-methionyl-L-methionine (DL-I) and D-methionyl-D-methionine (DD-I)
being unnatural (see scheme 1).
##STR00005##
[0042] In this connection, DD-I and LL-I are related to one another
as image and mirror image, i.e. they are enantiomers and thus have
the same physical properties. The same applies to the DL-I and LD-I
pair.
[0043] The two pairs DD/LL-I and DL/LD-I are by contrast
diastereomers of one another, i.e. they have different physical
data. Thus, for example, the DD/LL-I pair of diastereomers has a
solubility of 21.0 g/l in water at room temperature, whereas the
solubility of the DL/LD-I pair of diastereomers is 0.4 g/l.
[0044] Besides providing novel synthetic methods for preparing
methionylmethionine, the present invention, in a further
embodiment, provides a method employing DL-methionyl-DL-methionine
as feedstuff as DD/LL/DL/LD, DD/LL or DL/LD diastereomer mixture as
growth promoter for omnivorous, carnivorous and herbivorous fish
and crustaceans in aquacultures. According to an embodiment of the
present invention, DL-methionyl-DL-methionine (I) may be cleaved
under physiological conditions enzymatically by fish and
crustaceans to free D- and L-methionine (scheme 2) (see also
examples 22 to 24). For this purpose, the corresponding digestive
enzymes have been isolated from carp (omnivore), trout (carnivore)
and whiteleg shrimp (omnivore) and reacted with
DL-methionyl-DL-methionine in optimized in vitro experiments under
physiologically comparable conditions. The particular feature
according to the invention of the cleavage of
DL-methionyl-DL-methionine (I) is that all four possible
diastereomers, both the natural LL-I, and the three unnatural
diastereomers DD-, DL- and LD-I may be cleaved under physiological
conditions. This may apply both to the use of the complete mixture
of all diastereomers (DD/LL/DL/LD-I), and in each case to the two
pairs of diastereomers DD/LL-I and DL/LD-I (see FIG. 1).
##STR00006##
However, the cleavage of the individual diastereomers of
methionylmethionine takes place at different rates. This is
illustrated by the diagrammatic representation of the enzymatic
cleavage of the individual diastereomers of methionylmethionine
with digestive enzymes of fish and crustaceans in FIG. 2. However,
the delayed cleavage means that the liberation of D- and
L-methionine is likewise delayed (see FIG. 3). This has the great
advantage that there can be no fast-response absorption of free D-
or L-methionine in the digestive tract and thus no concentration
peak of free methionine in the blood plasma either.
[0045] The advantage of using methionylmethionine as feed additive
and methionine source according to the present invention may thus
be that D- or L-methionine is liberated in the body over the whole
digestion period and thus proceeds synchronously with the release
of other amino acids derived from natural protein sources
(slow-release mechanism) (see FIG. 3). This special effect results
in the simultaneous availability of all the important and essential
amino acids in an ideal ratio in the blood plasma being ensured, as
is absolutely necessary for an optimal growth of the body.
[0046] In the enzymatic cleavage of the DL-methionyl-DL-methionine
dipeptide (I), the unnatural D-methionine is also liberated in
addition to the natural L-methionine (see scheme 2). The former may
be enzymatically transaminated both by carnivorous, omnivorous and
herbivorous salt and fresh water fish and crustaceans to give
natural L-methionine. This is shown for the example of carp in
example 25. With the aid of an enzyme cocktail of digestive and
liver enzymes from carp, D-methionine may be transformed into
L-methionine under physiologically corresponding conditions (see
FIG. 4). An optimal supply of natural L-methionine to the body may
thus be ensured by use of DL-methionyl-DL-methionine (I).
[0047] The pelleting and extrusion experiments with various
mixtures of DL-methionyl-DL-methionine (I) and natural protein and
carbohydrate sources such as, for example, fish, corn and soybean
meal, and mixed with other essential amino acids, proteins,
peptides, vitamins, minerals, fats and oils, show that
DL-methionyl-DL-methionine (I) is absolutely stable during and
after the production process and no degradation or decomposition
whatsoever occurs (see example 26).
[0048] In order to investigate the leaching characteristics of the
diastereomers of methionylmethionine (I) from compound feed pellets
under water, the time-dependence of the dissolving out of
methionylmethionine was measured (see example 26). For comparison,
the leaching characteristics of DL-methionine, MHA and calcium-MHA
(MHA-Ca) were investigated under identical conditions. This study
shows that both the complete mixture of all the diastereomers
(DD/LL/DL/LD-I) and the pairs of diastereomers DD/LL-I and DL/LD-I
show distinctly less leaching than DL-methionine, MHA and
calcium-MHA (MHA-Ca) (see FIG. 5). Much less methionylmethionine is
thus dissolved out of the feed pellets over time than with all
other methionine derivatives. Particularly low leaching rates are
shown by the DL/LD-I pair of diastereomers, a maximum of only 5% of
which was dissolved out of the feed pellets even after a residence
time of 200 min (see FIG. 5).
[0049] A further preferred embodiment of the present invention
provides a process for preparing DL-methionyl-DL-methionine of
formula (I)
##STR00007##
by reacting a urea derivative of formula II
##STR00008##
wherein the radicals R.sup.1 and R.sup.2 in the urea derivatives
IIa, IIb, IIc, IId, IIe, IIf and IIg are defined as follows:
IIa: R.sup.1=COOH, R.sup.2=NHCONH.sub.2
IIb: R.sup.1=CONH.sub.2, R.sup.2=NHCONH.sub.2
IIc: R.sup.1=CONH.sub.2, R.sup.2=NH.sub.2
IId: R.sup.1--R.sup.2=--CONHCONH--
IIe: R.sup.1=CN, R.sup.2=OH
IIf: R.sup.1=CN, R.sup.2=NH.sub.2
IIg: R.sup.1==O, R.sup.2=H
[0050] to give DL-methionyl-DL-methionine (I).
[0051] In one embodiment of the process of the invention it is
moreover preferred for methioninehydantoin (IId) to be the starting
material or to be formed as intermediate product. In this process,
DL-methionyl-DL-methionine is synthesized directly from
methioninehydantoin and includes methods G, H, and J shown in
scheme 3.
##STR00009##
[0052] In a preferred embodiment of this method, a solution
comprising methioninehydantoin and water may be reacted with
methionine under basic conditions. It is further preferred for the
pH of the solution comprising the urea derivative to be adjusted to
a range from 8 to 14, preferably to from 9 to 13.5 and most
preferably from 10 to 13.
[0053] In a further preferred embodiment, the reaction takes place
at a temperature of from 50 to 200.degree. C., preferably at a
temperature of from 80 to 170.degree. C. and particularly
preferably at a temperature of from 130 to 160.degree. C.
[0054] It is further preferred for the reaction to be carried out
under pressure, preferably under a pressure of from 3 to 20 bar,
more preferably 4 to 18 bar and particularly preferably under a
pressure of from 6 to 15 bar.
[0055] In a further preferred embodiment of the process of the
present invention, a solution comprising methioninehydantoin and
water may be previously formed from one or more of the compounds
IIa, IIb, IIc, IId, IIe, IIf and IIg.
[0056] In another preferred embodiment of the process,
methioninehydantoin may be obtained by reacting the compound IIe or
IIf with a nitrogen-containing base, NH.sub.4HCO.sub.3,
(NH.sub.4).sub.2CO.sub.3, NH.sub.4OH/CO.sub.2 mixture or carbamate
salts. Reaction of the compound IIe may be preferably carried out
at a temperature of from 0.degree. C. to 150.degree. C., more
preferably 0.degree. C. to 100.degree. C. and particularly
preferably from 10.degree. C. to 70.degree. C.
[0057] In still another preferred embodiment of the process, the
methioninehydantoin is obtained by reacting the compound IIf with
CO.sub.2. In this embodiment, it is preferred that the reaction to
take place in the presence of a base, preferably selected from the
group comprising KHCO.sub.3, K.sub.2CO.sub.3, tertiary amines or
salts thereof, alkali metal and alkaline earth metal bases.
[0058] In an additional further preferred embodiment of the
process, methioninehydantoin is obtained by reacting the compound
IIg with a cyanide ion source and a base selected from the group
including nitrogen-containing bases, ammonium salts in the presence
of CO.sub.2, NH.sub.4HCO.sub.3, (NH.sub.4).sub.2CO.sub.3,
NH.sub.4OH/CO.sub.2 mixture and carbamate salts. The reaction in
this case takes place at a temperature of preferably -20.degree. C.
to 150.degree. C., preferably -10.degree. C. to 100.degree. C. and
particularly preferably from 0.degree. C. to 70.degree. C.
[0059] An alternative embodiment of the process of the invention
comprises:
a) reaction of the urea derivative of formulae IIa, IIb, IIc, IId,
IIe, IIf and IIg to give a diketopiperazine of the formula
(III)
##STR00010##
b) reaction of the diketopiperazine to give
DL-methionyl-DL-methionine. This process includes methods A, B, C
and D shown in scheme 3. In this process, diketopiperazine (III) is
formed as intermediate.
[0060] It is preferred in this embodiment, that the reaction of the
urea derivatives to give the diketopiperazine may be carried out at
a temperature of from 50.degree. C. to 200.degree. C., preferably
from 100.degree. C. to 180.degree. C. and particularly preferably
from 140.degree. C. to 170.degree. C. In a preferred embodiment of
this process, the reaction of the urea derivative to give the
diketopiperazine takes place under pressure, preferably under a
pressure of from 3 to 20 bar, more preferably 4 to 18 bar, and
particularly preferably under a pressure of from 6 to 15 bar.
[0061] The reaction of the urea derivative to give the
diketopiperazine preferably takes place in the presence of a base.
The base in this connection may be selected from the group of
nitrogen-containing bases, NH.sub.4HCO.sub.3,
(NH.sub.4).sub.2CO.sub.3, KHCO.sub.3, K.sub.2CO.sub.3,
NH.sub.4OH/CO.sub.2 mixture, carbamate salts, alkali metal and
alkaline earth metal bases. In a further preferred process, the
reaction of the urea derivative to give the diketopiperazine takes
place by reaction with methionine. A ratio of urea derivative to
methionine of from 1:100 to 1:0.5 may be preferred in this
embodiment.
[0062] In an additional further preferred process, the reaction of
the diketopiperazine to give DL-methionyl-DL-methionine takes place
by acidic hydrolysis. The acidic hydrolysis is in this case carried
out in the presence of an acid which is preferably selected from
the group of mineral acids, HCl, H.sub.2CO.sub.3,
CO.sub.2/H.sub.2O, H.sub.2SO.sub.4, phosphoric acids, carboxylic
acids and hydroxy carboxylic acids.
[0063] In another embodiment of the process of the invention, the
reaction of the diketopiperazine to give DL-methionyl-DL-methionine
may be by basic hydrolysis. In this case, the basic hydrolysis is
preferably carried out at a pH of from 7 to 14, particularly
preferably at a pH of from 9 to 12, very particularly preferably at
a pH of from 10 to 11, in order to obtain
DL-methionyl-DL-methionine. It may be moreover possible for the
basic conditions to be adjusted by using a substance which is
preferably selected from the group of nitrogen-containing bases,
NH.sub.4HCO.sub.3, (NH.sub.4).sub.2CO.sub.3, NH.sub.4OH/CO.sub.2
mixture, carbamate salts, KHCO.sub.3, K.sub.2CO.sub.3, carbonates,
alkali metal and alkaline earth metal bases.
[0064] The acidic or basic hydrolysis may preferably be carried out
at temperatures of from 50.degree. C. to 200.degree. C., preferably
from 80.degree. C. to 180.degree. C. and particularly preferably
from 90.degree. C. to 160.degree. C.
[0065] In a further embodiment, the reaction of the
diketopiperazine to give DL-methionyl-DL-methionine is carried out
by introducing CO.sub.2 into a basic solution, preferably into a
basic ammonium hydroxide, potassium hydroxide or sodium hydroxide
solution.
[0066] In a preferred embodiment of the process of the present
invention, the diketopiperazine may be isolated before the
hydrolysis. According to this embodiment, the diketopiperazine may
be isolated by crystallization from the reaction solution,
preferably at a temperature of from -30 to 120.degree. C., more
preferably at a temperature of 0 to 90.degree. C. and particularly
preferably at a temperature of from 10 to 70.degree. C.
[0067] To isolate the mixture of DD/LL/DL/LD-methionylmethionine
diastereomers from basic reaction solutions, the solutions are
acidified and the methionylmethionine may be obtained by
crystallization or precipitation. It is preferred that the
crystallization or precipitation be at a pH from 5 to 9,
particularly preferred for the pH to be from 5 to 7, and very
particularly preferred for the pH to be about 5.6. It may be
possible in this to employ acids preferably from the group of
mineral acids, HCl, H.sub.2CO.sub.3, CO.sub.2/H.sub.2O,
H.sub.2SO.sub.4, phosphoric acids, carboxylic acids and hydroxy
carboxylic acids for the acidification.
[0068] To isolate the mixture of DD/LL/DL/LD-methionylmethionine
diastereomers from acidic reaction solutions, bases may be added to
neutralize the reaction solutions, and the methionylmethionine may
be obtained by crystallization or precipitation. It is preferred in
this connection for the pH to be from 5 to 9, particularly
preferred for the pH to be from 5 to 7, and very particularly
preferred for the pH to be about 5.6. The bases used in this case
for the neutralization are preferably from the group of
NH.sub.4HCO.sub.3, (NH.sub.4).sub.2CO.sub.3, nitrogen-containing
bases, NH.sub.4OH, carbamate salts, KHCO.sub.3, K.sub.2CO.sub.3,
carbonates, alkali metal and alkaline earth metal bases.
[0069] In a special embodiment of the present invention, a process
for fractionating the mixture of DD/LL/DL/LD-methionylmethionine
diastereomers by fractional crystallization, thus obtaining the two
pairs of enantiomers DD/LL-methionylmethionine and
DL/LD-methionylmethionine is provided.
[0070] In a preferred embodiment of the process of fractional
crystallization of the present invention by acidification, the
procedure includes: acidification of the
DD/LL/DL/LD-methionylmethionine-containing suspension until a clear
solution is obtained; stepwise addition of a base to the acidic
solution until a precipitate of DL/LD-methionylmethionine is
obtained; and DD/LL-methionylmethionine is obtained from the mother
liquor. It is particularly preferred in this embodiment, for the
acidification to take place with an acid and for a pH of from 0.1
to 1.0, preferably a pH of about 0.6, to be set, and for the
resulting clear solution subsequently to be adjusted with a base to
a pH of from 5 to 6, preferably to a pH of about 5.6. It is
possible to use as acid in this connection mineral acids,
preferably phosphoric acid, sulfuric acid, hydrochloric acid or
carbonic acid, or carbon dioxide, and/or carboxylic acids,
especially the C.sub.1-C.sub.4 carboxylic acids formic acid, acetic
acid, propionic acid, butyric acid or isobutyric acid. Carbonic
acid or carbon dioxide may be particularly preferred as acids. It
may be possible according to this embodiment for the carbonic acid
or carbon dioxide to be introduced into the reaction mixture under
atmospheric pressure or under superatmospheric pressure.
[0071] The basic conditions are obtained by adding a base selected
from the group of consisting of NH.sub.4HCO.sub.3,
(NH.sub.4).sub.2CO.sub.3, nitrogen-containing bases, NH.sub.4OH,
carbamate salts, KHCO.sub.3, K.sub.2CO.sub.3, carbonates, alkali
metal bases and alkaline earth metal bases. In a further preferred
embodiment of the process of fractional crystallization by
basification, the procedure may comprise:
basification of the DD/LL/DL/LD-methionylmethionine-containing
suspension until a clear solution is obtained; stepwise addition of
an acid to the basic solution to obtain a precipitate of
DL/LD-methionylmethionine; removing the DL/LD-methionylmethionine
and obtaining DD/LL-methionylmethionine from the mother liquor.
[0072] It may be particularly preferred in this connection for the
basification to take place with a base and for a pH of from 7.5 to
14, preferably a pH of about 9 to 13, to beobtained, and for the
resulting clear solution subsequently to be adjusted with an acid
to a pH of from 5 to 6, preferably to a pH of about 5.6. Bases
preferably used in this case are bases from the group
NH.sub.4HCO.sub.3, (NH.sub.4).sub.2CO.sub.3, nitrogen-containing
bases, NH.sub.4OH, carbamate salts, KHCO.sub.3, K.sub.2CO.sub.3,
carbonates, alkali metal and alkaline earth metal bases.
[0073] The acidic conditions of the acidification are preferably
adjusted by using an acid from the group of mineral acids,
preferably phosphoric acid, sulfuric acid, hydrochloric acid, or
carbonic acid or carbon dioxide, and/or from the group of
carboxylic acids, in particular the C.sub.1-C.sub.4 carboxylic
acids formic acid, acetic acid, propionic acid, butyric acid and
isobutyric acid. Carbonic acid or carbon dioxide may be
particularly preferably used.
[0074] In a preferred embodiment of the process of fractional
crystallization, the temperature of the crystallization mixture is
from 0.degree. C. to 100.degree. C., preferably 5.degree. C. to
60.degree. C. and particularly preferably from 10.degree. C. to
40.degree. C.
[0075] The resulting DD/LL-methionylmethionine can moreover be
racemized and introduced into the separation process described
above, thus separating the two pairs of enantiomers
DD/LL-methionylmethionine and DL/LD-methionylmethionine from one
another.
[0076] All the processes of the present invention may be preferably
carried out in an aqueous medium.
[0077] The processes of the present invention may furthermore be
carried out in the batch process known to the skilled worker or in
continuous processes.
[0078] Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of illustration
only, and are not intended to be limiting unless otherwise
specified.
EXAMPLES
A) Overview of the Individual Steps and Methods of the Process of
the Invention
[0079] The process of the invention for preparing
DL-methionyl-DL-methionine (I) and the separation into the DD/LL-I
and DL/LD-I pairs of diastereomers are described in detail
below.
[0080] The process of the invention for preparing
DL-methionyl-DL-methionine (I) starts from a compound of the
general formula II
##STR00011##
where
IIa: R.sup.1=COOH, R.sup.2=NHCONH.sub.2
IIb: R.sup.1=CONH.sub.2, R.sup.2=NHCONH.sub.2
IIc: R.sup.1=CONH.sub.2, R.sup.2=NH.sub.2
IId: R.sup.1--R.sup.2=--CONHCONH--
IIe: R.sup.1=CN, R.sup.2=OH
IIf: R.sup.1=CN, R.sup.2=NH.sub.2
IIg: R.sup.1==O, R.sup.2=H.
[0081] This compound is transformed by various synthetic methods
(A, B, C, D, E, F, G, H and J) into DL-methionyl-DL-methionine (I)
(see scheme 3). In methods A, B, C, and D therein, the
corresponding diketopiperazine (III) is produced as intermediate.
In synthetic methods G, H and J, methionine hydantoin is produced
as intermediate and is transformed directly into
DL-methionyl-DL-methionine (I). It is subsequently possible by
fractional crystallization by method K to separate the two pairs of
diastereomers DD/LL-I and DL/LD-I (see scheme 3).
##STR00012##
B) Synthesis Examples
Example 1
Synthesis of 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) from N-carbamoylmethionine (IIa)
By Method A
[0082] 17.5 g (90.0 mmol, purity: 99%) of N-carbamoylmethionine
(IIa) were dissolved in 150 ml of water and stirred in a 200 ml
Roth steel autoclave with magnetic stirring at 160.degree. C. for 6
hours. The pressure increased during this period. From time to
time, gas was repeatedly discharged until a pressure of 7 bar was
reached. After completion of the reaction, the autoclave was cooled
in an ice bath. The resulting suspension was then filtered, and the
filtered solid was washed several times with water and dried in a
drying oven at 50.degree. C. in vacuo. The isolated yield was 8.1 g
(30.9 mmol) (69%) of bis[2-(methylthio)ethyl]-2,5-piperazinedione
(III), yellowish white crystals, purity>98% (HPLC), melting
point 234-236.degree. C.
[0083] .sup.1H-NMR of
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III) (500 MHz,
D.sub.6-DMSO): .delta.=1.85-2.05 (m, 4H,
2.times.SCH.sub.2CH.sub.2); 2.049 (s, 6H, 2.times.SCH.sub.3);
2.46-2.60 (m, 4H, 2.times.SCH.sub.2); 3.92-3.99 (m, 2H,
2.times.CH); 8.213 (s, 2H, 2.times.NH)
[0084] .sup.13C-NMR of
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III) (125.8 MHz,
D.sub.6-DMSO): .delta.=14.35 (CH.sub.3); 14.38 (CH.sub.3); 28.50
(CH.sub.2S); 28.68 (CH.sub.2S); 31.92 (CH.sub.2CH.sub.2S); 32.33
(CH.sub.2CH.sub.2S); 52.92 (CH); 52.96 (CH); 167.69 (C.dbd.O);
167.71 (C.dbd.O)
[0085] Elemental analysis for C.sub.10H.sub.18N.sub.2O.sub.2S.sub.2
(M=262.39 g/mol): Calculated: C 45.77; H 6.91; N 10.68; S 24.44
Found: C 45.94; H 6.96; N 10.64; S 24.38
Example 2
Synthesis of 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) from
2-[(aminocarbonyDamino]-4-(methylthio)butanoamide
(N-carbamoylmethioninamide) (IIb) By Method A
[0086] 17.4 g (90.0 mmol, purity: 98.5%) of
2-[(aminocarbony)amino]-4-(methylthio)butanoamide (IIb) were
dissolved in 150 ml of water and stirred in a 200 ml Roth steel
autoclave with magnetic stirring at 160.degree. C. for 7 hours. The
pressure increased during this heating. From time to time, gas was
repeatedly discharged until a pressure of 7 bar was reached. After
completion of the reaction, the autoclave was cooled in an ice
bath. The resulting suspension was then filtered, and the filtered
solid was washed several times with water and dried in a drying
oven at 50.degree. C. in vacuo. The isolated yield was 9.2 g (35.1
mmol) (78%) of bis[2-(methylthio)ethyl]-2,5-piperazinedione (III),
yellowish white crystals, purity>98% (HPLC).
[0087] The melting point and the NMR data agreed with those of
example 1.
Example 3
Synthesis of 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) from
5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (IId)
(methioninehydantoin) By Method A and Subsequent Reuse of the
Mother Liquor (Cascade Reaction)
[0088] First Batch:
[0089] A suspension of 13.4 g (0.09 mol) of methionine, 17.2 g
(0.09 mol, purity: 91%) of methioninehydantoin (IId) and 150 g of
water were introduced into a 200 ml Roth steel autoclave with
magnetic stirring and stirred at 160.degree. C. for 6 hours, during
which the pressure increased to 15 bar. From time to time, the
autoclave was decompressed until the pressure settled at a constant
10 bar. The autoclave was then cooled in an ice bath, and the
resulting suspension was filtered and the solid was washed with 75
ml of water. Finally, the solid was dried in a vacuum drying oven
at 50.degree. C. overnight.
Bis[2-(methylthio)ethyl]-2,5-piperazinedione (III) was isolated as
yellowish white crystals.
[0090] Subsequent Batches:
[0091] The washing water and the mother liquor from the preceding
batch were combined and concentrated to 90 ml in a rotary
evaporator at 50.degree. C. 17.2 g (0.09 mol, purity: 91%) of
methioninehydantoin (IId) were taken up with the concentrated
mother liquor and made up to 150 g of solution with water. The
resulting solution was introduced into a 200 ml Roth steel
autoclave with magnetic stirring and stirred at 160.degree. C. for
6 hours, during which the pressure increased to 15 bar. From time
to time, the autoclave was decompressed until the pressure remained
constant at 10 bar. Further working up took place as described for
the first batch.
Example 4
Synthesis of 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) from
2-amino-4-(methylthio)butanoamide (methioninamide) (IIc) By Method
B
[0092] 16.6 g (0.09 mol) of 2-amino-4-(methylthio)butanoamide
hydrochloride (IIc) and 8.7 g (0.09 mol) of
(NH.sub.4).sub.2CO.sub.3 were dissolved in 150 g of water and
stirred in a 200 ml Roth steel autoclave with magnetic stirring at
160.degree. C. for 6 hours. The autoclave was then cooled in an ice
bath. The resulting suspension was then filtered, and the filtered
solid was washed several times with water and dried in a drying
oven at 50.degree. C. in vacuo. The isolated yield was 6.5 g (24.8
mmol) (55%) of bis[2-(methylthio)ethyl]-2,5-piperazinedione (III),
yellowish white crystals, purity>98% (HPLC).
[0093] The melting point and the NMR data agreed with those from
example 1.
Example 5
Synthesis of 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) from
2-hydroxy-4-(methylthio)butanenitrile
(3-(methylmercapto)propionaldehyde cyanohydrin, MMP-CH) (IIe) By
Method C
[0094] A solution of 30.5 g (0.232 mol) of
2-hydroxy-4-(methylthio)butanenitrile (IIe) and 360 g of water was
slowly added dropwise at RT to a suspension of 22.4 g (0.283
mol=1.22 eq.) of NH.sub.4HCO.sub.3 in 20 g of water and stirred for
2 h. The NH.sub.4HCO.sub.3 dissolved during this time. The
resulting solution was subsequently stirred at 50.degree. C. for 7
h and then at room temperature overnight. The reaction mixture was
then transferred into a 500 ml steel autoclave, heated to
160.degree. C., and stirred at this temperature for 6 hours. The
autoclave was then cooled in an ice bath, the resulting suspension
was filtered, and the solid was washed with 50 ml of water.
Finally, the pale solid was dried in a vacuum drying oven at
50.degree. C. overnight. The isolated yield was 17.8 g (67.8 mmol)
(58%) of bis[2-(methylthio)ethyl]-2,5-piperazinedione (III),
yellowish white crystals, purity>98% (HPLC).
[0095] The melting point and the NMR data agreed with those from
example 1.
Example 6
Synthesis of 3 ,6-bis[2-(methylthio)ethyl]-2,5 -piperazinedione
(III) (methioninediketopiperazine, DKP) From
2-amino-4-(methylthio)butanenitrile (methioninenitrile) (IIf) By
Method C
[0096] A moderate stream of CO.sub.2 was passed into a solution of
26.2 g (0.201 mol) of 2-amino-4-(methylthio)butanenitrile (IIf) in
330 g of water over a period of 3 hours, during which the
temperature rose to 45.degree. C. and the pH settled at 8. Stirring
was then continued at room temperature overnight. The next morning,
the reaction mixture was transferred into a 500 ml steel autoclave,
heated to 160.degree. C. and stirred at this temperature for 6
hours. The autoclave was then cooled in an ice bath, the resulting
suspension was filtered, and the solid was washed with 50 ml of
water and dried in a vacuum drying oven at 50.degree. C. overnight.
The isolated yield was 15.7 g (59.7 mmol) (59%) of
bis[2-(methylthio)ethyl]-2,5-piperazinedione (III), yellowish white
crystals, purity>98% (HPLC).
[0097] The melting point and the NMR data agreed with those from
example 1.
Example 7
Synthesis of 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) From 3-(methylthio)propanaldehyde
(3-(methylmercapto)propionaldehyde, MMP) (IIg) By Method D
[0098] 66.0 g (0.68 mol) of (NH.sub.4).sub.2CO.sub.3 were
introduced into 100 g of water and cooled to 5.degree. C. in an ice
bath. Then, over the course of 25 minutes, 16.6 g (0.61 mol) of
freshly distilled hydrocyanic acid were added dropwise, during
which the temperature of the suspension was kept in the range from
5 to 10.degree. C. Addition of 860 g of water was followed by
dropwise addition, at 10.degree. C., of 60.3 g (0.58 mol) of
3-(methylthio)propionaldehyde (IIg) over a period of 80 min. The pH
remained constant in the range from 8.5 to 9 during this. The
reaction mixture was then heated to 50.degree. C. and stirred at
this temperature for 7 hours. After completion of the reaction, the
reaction mixture was cooled to 5.degree. C. in an ice bath and
stored in a refrigerator overnight. The next morning, the mixture
was transferred into a 21 steel autoclave, heated to 160.degree. C.
and stirred at this temperature for 6 hours. The autoclave was then
cooled in an ice bath, the resulting suspension was filtered and
washed with 150 ml of water, and the solid was dried in a vacuum
drying oven at 50.degree. C. overnight. The isolated yield was 48.6
g (185.2 mmol) (64%) of
bis[2-(methylthio)ethyl]-2,5-piperazinedione (III), yellowish white
crystals, purity>98% (HPLC).
[0099] The melting point and the NMR data agreed with those from
example 1.
Example 8
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) with Concentrated Hydrochloric
Acid By Method E
[0100] 655.9 g (2.50 mol) of
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III) (DKP) were
suspended in 1661 g of water. While stirring, 271.0 g of conc.
hydrochloric acid were very slowly added dropwise and then
cautiously heated, with very vigorous stirring, to reflux. Severe
foaming may occur during this. The reaction mixture was heated to
reflux for 5.5 hours, thus dissolving all the solid. During the
subsequent cooling, unreacted DKP (III) precipitated and was
filtered off. This DKP may be employed again for further hydrolyses
in later reactions. The filtrate was then adjusted to pH 6 in a
glass beaker in an ice bath with 32% strength aqueous ammonia. A
DD/LL/DL/LD-methionylmethionine (I) separates out as a thick mass
of crystals, and 50:50 mixture of the two pairs of diastereomers
(DL/LD-Met-Met) (DL/DL-I) and (DD/LL-Met-Met) (DD/LL-I) during
this. It was finally dried in a drying oven at 60.degree. C. in
vacuo. Yield: 601.0 g (2.14 mol) (85.7%) of
DD/LL/DL/LD-methionylmethionine (I), slightly yellowish solid,
purity 98% (HPLC).
[0101] .sup.1H-NMR of DD/LL/DL/LD-methionylmethionine (I) (500 MHz,
D.sub.6-DMSO+HCl): .delta.=1.86-2.16 (m, 4H,
2.times.SCH.sub.2CH.sub.2); 2.050 (s, 3H, SCH.sub.3); 2.060 (s, 3H,
SCH.sub.3); 2.44-2.64 (m, 4H, 2.times.SCH.sub.2); 2.90-4.00 (m, 1H,
CH); 4.32-4.42 (m, 1H, CH); 8.45 (bs, 3H, NH.sub.3.sup.+);
8.98-9.08 (m, 1H, 2.times.NH)
[0102] .sup.13C-NMR of DD/LL/DL/LD-methionylmethionine (I) (125.8
MHz, D.sub.6-DMSO+HCl): .delta.=14.33 (CH.sub.3); 14.38 (CH.sub.3);
27.74; 27.94; 29.51; 30.04; 30.13; 30.89; 30.95; 51.00; 51.29;
51.54 (CH, CH.sub.2); 168.05 (CONH); 168.19 (CONH); 172.55 (COOH);
172.62 (COOH)
[0103] Elemental analysis for C.sub.10H.sub.20N.sub.2O.sub.3S.sub.2
(M=280.41 g/mol): Calculated: C 42.83; H 7.19; N 9.99; S 22.87
Found: C 42.61; H 7.19; N 10.06; S 22.72
Example 9
Industrial synthesis of DD/LL/DL/LD-methionylmethionine (I) from
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) With Concentrated Hydrochloric
Acid By Method E
[0104] 500 l of water were introduced into a 500 l enameled tank
with stirrer, 32 l of concentrated hydrochloric acid and 78.6 kg of
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III) (DKP) were
added, and the apparatus was closed tightly. It was then heated at
110.degree. C. while stirring for 2 hours, during which the
pressure rose to 2.5 bar and the DKP (III) virtually completely
dissolved. After the reaction was complete, the mixture was cooled
to 20.degree. C., and the unreacted DKP was spun down in a
centrifuge. The solid was washed with 10 l of water. The filtrate
and washing water were then collected in an 800 l container and
subsequently introduced into a 500 l tank with stirrer again.
Addition of 2 kg of activated carbon was followed by stirring at
20.degree. C. for 30 min. The suspension was then filtered through
a filter press into a further 500 l tank with stirrer. About 28 l
of concentrated ammonia solution were then added to precipitate at
pH 6 the DD/LL/DL/LD-methionylmethionine (I). During this there was
an initial preferential precipitation of the less soluble racemic
pair of diastereomers DL/LD-methionylmethionine (DL/LD-I). This was
spun down and the mother liquor was concentrated together with
washing water to one quarter of the original volume in vapor pump
vacuum at an internal temperature not exceeding 40.degree. C.
During this, the more soluble racemic pair of diastereomers
DD/LL-methionylmethionine (DD/LL-I) crystallized together with
small amounts of the slightly soluble DL/LD-I. Completion of the
distillation was followed by cooling to 20.degree. C. and
centrifugation. The separated mother liquor and washing water were
discarded. Both fractions were dried in vacuo at 70.degree. C. In
total, it was possible to obtain 64.2 kg (78%) of
DD/LL/DL/LD-methionyl-methionine (I) as mixture of diastereomers.
Purity>98% (HPLC).
[0105] The melting point and the NMR data agreed with those from
example 8.
Example 10
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) Under Alkaline Conditions, e.g.
With Ammonia By Method F
[0106] 65.6 g (0.25 mol) of
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III) (DKP), 70 ml
of 25% strength ammonia solution and 500 ml of water were heated at
150.degree. C. in an autoclave for 2 hours. After cooling, the
unreacted DKP (III) (16.0 g=24.4%) was filtered off with suction.
This can be employed again in a subsequent batch. The filtrate was
concentrated in a rotary evaporator at a water temperature of
80-90.degree. C. until the first crystals separated out. After
cooling and leaving to stand overnight it was possible to isolate
after filtration and drying in total 49.3 g (70.3%) of
DD/LL/DL/LD-methionylmethionine (I) as 50:50 mixture of the two
pairs of diastereomers DL/DL-I and DD/LL-I as a white solid. Purity
98% (HPLC).
[0107] The melting point and the NMR data agreed with those from
example 8.
Example 11
Purification of DD/LL/DL/LD-methionylmethionine (I)
[0108] 500 g of DD/LL/DL/LD-methionylmethionine (I) were suspended
in 7800 g of deionized water (pH 5.3). At 26.degree. C., the pH was
adjusted to 1.0 with 346.6 g of 50% by weight sulfuric acid. The
methionylmethionine dissolved completely. For clarification, 18 g
of activated carbon were added to the yellowish turbid solution and
stirred for 60 minutes. The activated carbon was filtered off, and
the clear colorless solution was adjusted to pH 5.6 with 228 g of
32% by weight ammonia solution. The solution was left to stand
overnight. The precipitated white solid was filtered off with
suction and dried in a drying oven at 50.degree. C. in vacuo.
Yield: 460.5 g (92%) of DD/LL/DL/LD-methionylmethionine (I),
brilliant white solid, purity>99% (HPLC).
[0109] The NMR data agreed with those from example 8.
Example 12
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
N-carbamoylmethionine (IIa) and DL-methionine with KOH By Method
G
[0110] 13.4 g (0.09 mol) of DL-methionine, 17.5 g (0.09 mol,
purity: 99%) of N-carbamoylmethionine (IIa) and 11.9 g (0.18 mol)
of 85% pure KOH were dissolved in 150 ml of water and stirred at
150.degree. C. in a 200 ml Roth steel autoclave with magnetic
stirring for 5 hours, during which the pressure increased to 6 bar.
After reaction was complete, the autoclave was cooled, and the
precipitated 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) was filtered off and washed with
a little water. The washing water and the mother liquor were
combined and concentrated to a volume of 130 ml in a rotary
evaporator at 40.degree. C. A moderate stream of CO.sub.2 was then
passed into the resulting solution until a pH of 6.4 was reached
and a white solid precipitated. This was filtered off, washed with
a little cold water and dried in a vacuum drying oven at 50.degree.
C. overnight. The isolated yield was 11.4 g (40.6 mmol) (45%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity >98%
(HPLC).
[0111] The NMR data agreed with those from example 8.
Example 13
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (IId)
(methioninehydantoin) and DL-methionine with KOH By Method G
[0112] 13.4 g (0.09 mol) of DL-methionine, 17.2 g (0.09 mol,
purity: 91%) of methioninehydantoin (IId) and 8.9 g (0.135 mol) of
85% pure KOH were dissolved in 150 ml of water and stirred at
150.degree. C. in a 200 ml Roth steel autoclave with magnetic
stirring for 5 hours, during which the pressure increased to 8 bar.
After the reaction was complete, the autoclave was cooled, the
resulting suspension was filtered and the precipitated
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) was washed several times with a
little water. Mother liquor and washing water were combined, and
the resulting solution was concentrated to a volume of 125 ml in a
rotary evaporator at 40.degree. C. The concentrate was cautiously
neutralized with concentrated hydrochloric acid. A white solid
precipitated on stirring at room temperature and at a pH of 5.8
overnight. This solid was filtered off, washed with a little cold
water and dried in a vacuum drying oven at 50.degree. C. overnight.
The isolated yield was 17.5 g (62.4 mmol) (69%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity>98%
(HPLC).
[0113] The NMR data agreed with those from example 8.
Example 14
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (IId)
(methioninehydantoin) and DL-methionine with K.sub.2CO.sub.3 By
Method G
[0114] 13.4 g (0.09 mol) of DL-methionine, 17.2 g (0.09 mol,
purity: 91%) of methioninehydantoin (IId) and 12.4 g (0.09 mol) of
K.sub.2CO.sub.3 were dissolved in 150 ml of water and stirred at
150.degree. C. in a 200 ml Roth steel autoclave with magnetic
stirring for 5 hours, during which the pressure increased to 12
bar. After the reaction was complete, the autoclave was cooled, and
the precipitated 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione
(III) (methioninediketopiperazine, DKP) was filtered off and washed
with a little water. The washing water and the mother liquor were
combined and concentrated to a volume of 135 ml in a rotary
evaporator at 40.degree. C. A moderate stream of CO.sub.2 was then
passed into the resulting solution until a pH of 6.8 was reached
and a white solid precipitated. This was filtered off, washed with
a little cold water and dried in a vacuum drying oven at 50.degree.
C. overnight. Yield: 14.3 g (60.0 mmol) (57%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity>99%
(HPLC).
[0115] The NMR data agreed with those from example 8.
Example 15
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
5-[2-(methylthio)ethyl]-2,4-imidazolidinedione (IId)
(methioninehydantoin) and DL-methionine with KHCO.sub.3 by Method
G
[0116] 13.4 g (0.09 mol) of DL-methionine, 17.2 g (0.09 mol,
purity: 91%) of methioninehydantoin (IId) and 9.1 g (0.09 mol) of
KHCO.sub.3 were dissolved in 150 ml of water and stirred at
150.degree. C. in a 200 ml Roth steel autoclave with magnetic
stirring for 5 hours, during which the pressure increased to 12
bar. After the reaction was complete, the autoclave was cooled, and
the precipitated 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione
(III) (methioninediketopiperazine, DKP) was filtered off and washed
with a little water. The washing water and the mother liquor were
combined and concentrated to a volume of 120 ml in a rotary
evaporator at 40.degree. C. A moderate stream of CO.sub.2 was then
passed into the resulting solution until a pH of 6.3 was reached
and a white solid precipitated. This was filtered off, washed with
a little cold water and dried in a vacuum drying oven at 50.degree.
C. overnight. Yield: 16.0 g (57.1 mmol) (63%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity>99%
(HPLC).
[0117] The NMR data agreed with those from example 8.
Example 16
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
2-amino-4-(methylthio)butanoamide (IIc) (methioninamide) and
DL-methionine with (NH.sub.4).sub.2CO.sub.3 By Method H
[0118] 8.3 g (0.045 mol) of 2-amino-4-(methylthio)butanoamide (IIc)
hydrochloride, 6.7 g (0.045 mol) of methionine, 4.3 g (0.045 mol)
of (NH.sub.4).sub.2CO.sub.3 and 3.0 g (0.045 mol) of 85% pure KOH
were dissolved in 75 g of water and stirred at 160.degree. C. in a
200 ml Roth steel autoclave with magnetic stirring for 6 hours. The
autoclave was then cooled in an ice bath, the resulting suspension
was filtered off, and the precipitated
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) was washed with a little water.
The washing water and the mother liquor were combined and
concentrated to a volume of 70 ml in a rotary evaporator at
40.degree. C. A moderate stream of CO.sub.2 was then passed into
the resulting solution until a pH of 6.3 was reached and a white
solid precipitated. This was filtered off, washed with a little
cold water and dried in a vacuum drying oven at 50.degree. C.
overnight. Yield: 7.8 g (27.8 mmol) (62%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity>98%
(HPLC).
[0119] The NMR data agreed with those from example 8.
Example 17
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
2-hydroxy-4-(methylthio)butanenitrile (IIe)
(3-(methylmercapto)propionaldehyde cyanohydrin, MMP-CH) and
DL-methionine with NH.sub.4HCO.sub.3 By Method H
[0120] 15.2 g (0.116 mol) of 2-hydroxy-4-(methylthio)butanenitrile
(IIe) were slowly added dropwise at RT to a suspension of 11.1 g
(0.141 mol=1.22 eq.) of NH.sub.4HCO.sub.3 in 10 g of water and
stirred for 2 h. The NH.sub.4HCO.sub.3 dissolved during this. Then
180 g of water were added and the resulting solution was stirred at
50.degree. C. for 7 h and at room temperature overnight. The next
morning, 17.3 g (0.116 mol) of methionine, 7.7 g (0.116 mol) of 85%
pure KOH and a further 180 g of water were added, and the reaction
mixture was transferred into a 11 steel autoclave, heated to
160.degree. C. and stirred at this temperature for 6 hours. The
autoclave was then cooled in an ice bath, the resulting suspension
was filtered, and the precipitated
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) was washed with 100 ml of water.
Mother liquor and washing water were combined, and the resulting
solution was concentrated to a volume of 160 ml in a rotary
evaporator at 40.degree. C. The concentrate was cautiously
neutralized with 50% strength sulfuric acid. A white solid
precipitated on stirring at room temperature and at a pH of 5.4
overnight. This solid was filtered off, washed with a little cold
water and dried in a vacuum drying oven at 50.degree. C. overnight.
Yield: 15.2 g (54.2 mmol) (47%) of DD/LL/DL/LD-methionylmethionine
(I), white solid, purity>99% (HPLC).
[0121] The NMR data agreed with those from example 8.
Example 18
Synthesis of DD/LL/DL/LD-methionylmethionine (I) from
2-amino-4-(methylthio)butanenitrile (IIf) (methioninenitrile) with
CO.sub.2 and DL-methionine By Method H
[0122] A moderate stream of CO.sub.2 was passed into a solution of
26.2 g (0.201 mol) of 2-amino-4-(methylthio)butanenitrile (IIf) in
330 g of water over a period of 3 hours, during which the
temperature rose to 45.degree. C. and the pH settled at 8. Stirring
was then continued at room temperature overnight. The next morning,
the reaction mixture was mixed with 30.0 g (0.201 mol) of
methionine and 13.3 g (0.201 mol) of 85% pure KOH and transferred
into a 1 l steel autoclave, heated to 160.degree. C. and stirred at
this temperature for 6 hours. The autoclave was then cooled in an
ice bath, the resulting suspension was filtered, and the
precipitated 3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) was washed with a little water.
The washing water and the mother liquor were combined and
concentrated to a volume of 280 ml in a rotary evaporator at
40.degree. C. A moderate stream of CO.sub.2 was then passed into
the resulting solution until a pH of 6.0 was reached and a white
solid precipitated. This was filtered off, washed with a little
cold water and dried in a vacuum drying oven at 50.degree. C.
overnight. Yield: 32.7 g (116.6 mmol) (58%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity >98%
(HPLC).
[0123] The NMR data agreed with those from example 8.
Example 19
Synthesis of DD/LL/DL/LD-methionylmethionine (I) From
3-(methylthio)propanaldehyde (IIg) (MMP) with Hydrocyanic Acid,
Ammonium Carbonate and DL-methionine By Method J
[0124] 66.0 g (0.68 mol) of (NH.sub.4).sub.2CO.sub.3 were
introduced into 100 g of water and cooled to 5.degree. C. in an ice
bath. Then 16.55 g (0.612 mol) of freshly distilled hydrocyanic
acid were added dropwise over the course of 25 min, during which
the temperature of the suspension was kept at 5 to 10.degree. C.
After 500 g of water had been added, 60.3 g (0.58 mol) of
3-(methylthio)propionaldehyde (IIg) were added dropwise at
10.degree. C. over a period of 80 min. The pH remained constant in
the range from 8.5 to 9 during this. The reaction mixture was then
heated to 50.degree. C. and stirred at this temperature for 7
hours. After the reaction was complete, the reaction mixture was
cooled to 5.degree. C. in an ice bath and stored in a refrigerator
overnight. The next morning, 86.5 g (0.58 mol)
2-amino-4-(methylthio)butanoic acid (methionine), 38.3 g (0.58 mol)
of 85% pure KOH (0.58 mol), and a further 530 g of water were
added. The mixture was transferred into a 21 steel autoclave,
heated to 160.degree. C. and stirred at this temperature for 6
hours. The autoclave was then cooled in an ice bath, the resulting
suspension was filtered, and the precipitated
3,6-bis[2-(methylthio)ethyl]-2,5-piperazinedione (III)
(methioninediketopiperazine, DKP) was washed with a little water.
The washing water and the mother liquor were combined and
concentrated to a volume of 800 ml in a rotary evaporator at
40.degree. C. A moderate stream of CO.sub.2 was then passed into
the resulting solution until a pH of 6.0 was reached and a white
solid precipitated. This was filtered off, washed with a little
cold water and dried in a vacuum drying oven at 50.degree. C.
overnight. Yield: 85.1 g (0.30 mol) (52%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity>98%
(HPLC).
[0125] The NMR data agreed with those from example 8.
Example 20
Separation of the Two Pairs of Diastereomers
DD/LL-methionylmethionine (DD/LL-I) and DL/LD-methionylmethionine
(DL/LD-I) by Fractional Crystallization from
DD/LL/DL/LD-methionylmethionine (I) by Method K
[0126] a) DL/LD-Methionylmethionine (DL/LD-I):
[0127] 290.4 g of DD/LL/DL/LD-methionylmethionine (I) (50:50
mixture of DD/LL-I and DL/LD-I) were suspended in 2614 g of
deionized water and adjusted to pH 0.6 with 381.7 g of 50% by
weight sulfuric acid. The clear colorless solution was adjusted to
pH 5.6 with 265.9 g of 32% by weight ammonia solution, and the
resulting white precipitate was filtered off with suction (580.9 g
moist). The solid was finally dried in a drying oven in vacuo at
50.degree. C. The yield was 126.2 g (86.9%) of
DL/LD-methionylmethionine (DL/LD-I), white solid, purity>98%
(HPLC), melting range 232-233.degree. C. (decomp.).
[0128] .sup.1H-NMR of DL/LD-methionylmethionine (DL/LD-I) (500 MHz,
D.sub.6-DMSO+HCl): 1.88-2.12 (m, 4H, 2.times.SCH.sub.2CH.sub.2);
2.031 (s, 3H, CH.sub.3); 2.041 (s, 3H, CH.sub.3); 2.48-2.56 (m, 4H,
2.times.SCH.sub.2); 3.87-3.95 (m, 1H, CH); 4.30-4.38 (m, 1H, CH);
8.429 (d, 3H, .sup.3J=4.4 Hz, NH.sub.3.sup.+); 9.034 (d, 1H,
.sup.3J=8.0 Hz, NH)
[0129] .sup.13C-NMR of DL/LD-methionylmethionine (DL/LD-I) (125.8
MHz, D.sub.6-DMSO+HCl): 14.57 (CH.sub.3); 14.62 (CH.sub.3); 28.19;
29.75; 30.28; 31.19; 51.25 (CH); 51.79 (CH); 168.29 (CONH); 172.80
(COOH)
[0130] Solubility (water, 20.degree. C.): 0.4 g/l
b) DD/LL-Methionylmethionine (DD/LL-I):
[0131] The colorless mother liquor from a) was concentrated in a
rotary evaporator at 35.degree. C. under water pump vacuum. A white
suspension was obtained. The white solid composed of ammonium
sulfate, residues of DL/LD-I and target compound was then filtered
off with suction and dried in vacuo at 50.degree. C. The three
solids were separated by suspending the mixture in deionized water
and stirring. The undissolved DL/LD-I was filtered off with
suction, and the mother liquor was concentrated to about one fifth
in a rotary evaporator at 50.degree. C. under water pump vacuum.
After prolonged standing, DD/LL-methionylmethionine (DD/LL-I)
crystallized as a white solid. It was finally filtered off with
suction and dried in a vacuum drying oven at 50.degree. C. The
yield was 78.2 g (53.9%) based on DD/LL-methionylmethionine
(DD/LL-I), white solid, >96% (HPLC), melting range
226-227.degree. C. (decomposition).
[0132] .sup.1H-NMR of DD/LL-methionylmethionine (DD/LL-I) (500 MHz,
D.sub.6-DMSO+HCl): 1.84-2.12 (m, 4H, 2.times.SCH.sub.2CH.sub.2);
2.044 (s, 3H, CH.sub.3); 2.046 (s, 3H, CH.sub.3); 2.48-2.62 (m, 4H,
2.times.SCH.sub.2); 3.89-3.97 (m, 1H, CH); 4.33-4.40 (m, 1H, CH);
8.422 (d, 3H, .sup.3J=4.0 Hz, NH.sub.3.sup.+); 9.065 (d, 1H,
.sup.3J=7.5 Hz, NH)
[0133] .sup.13C-NMR of DD/LL-methionylmethionine (DD/LL-I) (125.8
MHz, D.sub.6-DMSO+HCl): 14.56 (CH.sub.3); 14.57 (CH.sub.3); 27.97;
29.73; 30.35; 31.11; 51.22 (CH); 51.50 (CH); 168.41 (CONH); 172.83
(COOH)
[0134] Solubility (water, 20.degree. C.): 21.0 g/l
Example 21
Racemization of the Two Pairs of Diastereomers
DD/LL-methionylmethionine (DD/LL-I) and DL/LD-methionylmethionine
(DL/LD-I) Under Basic Conditions
[0135] a) Racemization of DL/LD-methionylmethionine (DL/LD-I)
[0136] 12.6 g (45.0 mmol) of the pair of diastereomers
DL/LD-methionylmethionine (DL/LD-I) were dissolved together with
3.1 g (22.5 mmol) of K.sub.2CO.sub.3 in 75 ml of water in a 200 ml
Roth laboratory reactor and heated to 160.degree. C. while
stirring. The pressure rose to 7 bar during this. After 6 hours at
this temperature, the autoclave was cooled in an ice bath. The
resulting suspension was then filtered, and the solid was filtered
off, washed several times with water and dried in a drying oven in
vacuo at 50.degree. C. The isolated yield was 6.5 g (24.8 mmol)
(55%) of bis[2-(methylthio)ethyl]-2,5-piperazinedione (III),
yellowish white crystals, purity>98%, melting point
234-236.degree. C.; diastereomer ratio: 52:48 (DD/LL-III:meso-III).
The washing water and the mother liquor were combined and
concentrated to a volume of 25 ml in a rotary evaporator at
40.degree. C. A moderate stream of CO.sub.2 was then passed into
the resulting solution until the pH reached 6.0 and a white solid
precipitated. This was filtered off, washed with a little cold
water and dried in a vacuum drying oven at 50.degree. C. overnight.
The isolated yield was 5.7 g (20.3 mmol) (45%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity>98%
(HPLC).
[0137] The NMR data agreed with those from example 8.
[0138] b) Racemization of DD/LL-methionylmethionine (DD/LL-I)
[0139] 12.6 g (45.0 mmol) of DD/LL-methionylmethionine (DD/LL-I)
were dissolved together with 4.5 g (45.0 mmol) of KHCO.sub.3 in 75
ml of water in a 200 ml Roth laboratory reactor and heated to
160.degree. C. while stirring. The pressure increased to 7 bar and,
after 6 hours at this temperature, the autoclave was cooled in an
ice bath. The resulting suspension was then filtered, and the
filtered solid was washed several times with water and dried in a
drying oven in vacuo at 50.degree. C. The isolated yield was 6.0 g
(22.9 mmol) (51%) of bis[2-(methylthio)ethyl]-2,5-piperazinedione
(III), yellowish white crystals, purity>98% (HPLC), melting
point 233-236.degree. C.; diastereomer ratio: 54:46 (DD/LL-III :
meso-III). The washing water and the mother liquor were combined
and concentrated to a volume of 25 ml in a rotary evaporator at
40.degree. C. A moderate stream of CO.sub.2 was then passed into
the resulting solution until the pH reached 6.0 and a white solid
precipitated. This was filtered off, washed with a little cold
water and dried in a vacuum drying oven at 50.degree. C. overnight.
The isolated yield was 5.5 g (19.6 mmol) (44%) of
DD/LL/DL/LD-methionylmethionine (I), white solid, purity>98%
(HPLC).
[0140] The NMR data agreed with those from example 8.
Example 22
[0141] In Vitro Digestion Experiments on DL-methionyl-DL-methionine
(I) With Digestive Enzymes From Omnivorous Carp
[0142] a) Isolation of the Digestive Enzymes From Common Carp
(Cyprinus carpio morpha noblis)
[0143] The method for isolating the digestive enzymes was based on
that of EID and MATTY (Aquaculture 1989, 79, 111-119). For this
purpose, the intestine of five one-year old common carp (Cyprinus
carpio morpha noblis) was exposed, rinsed with water and cut open
longitudinally, and in each case the intestinal mucosa was scraped
off. This was comminuted together with crushed ice using a mixer.
The resulting suspension was treated with an ultrasonic probe in
order to disrupt cells which were still intact. The cell
constituents and fat were separated by centrifuging the suspension
at 4.degree. C. for 30 minutes, and the homogenate was decanted off
and sterilized with a trace of thimerosal. 260.7 ml of enzyme
solution from the intestinal mucosa were obtained from 5 common
carp, and the solution was stored in the dark at 4.degree. C.
[0144] b) Procedure for the In Vitro Digestion Investigations
[0145] DL-Methionyl-DL-methionine (I) and the corresponding pairs
of diastereomers DD/LL-I and DL/LD-I were taken up in TRIS/HCl
buffer solution and mixed with the enzyme solution. A blank was
made up in each case without enzyme solution for comparison and to
estimate the purely chemical cleavage rate. A sample was taken from
time to time, and the composition thereof was detected and
quantified with the aid of a calibrated HPLC. The conversion was
determined as the quotient of the area for methionine and the area
for methionylmethionine (I) (see FIGS. 6 and 7).
TABLE-US-00001 TABLE 1 Sample Blank Precharge Substrate 0.143 mmol
0.143 mmol Met-Met (I) (40.1 mg) (40.1 mg) TRIS/HCl buffer
solution, 5.7 ml 8.3 ml pH 9.5 Reaction start Enzyme solution 2.6
ml -- ({circumflex over (=)} 5% carp solution) Reaction 37.degree.
C. 37.degree. C. Reaction stop 0.2 ml of reaction solution was
taken up in 9.8 ml of 10% strength H.sub.3PO.sub.4 solution.
Example 23
In Vitro Digestion Experiment on DL-methionyl-DL-methionine (I)
with Digestive Enzymes From Carnivorous Trout
[0146] a) Isolation of the Digestive Enzymes From Rainbow Trout
(Oncorhynchus mykiss)
[0147] The method for isolating the digestive enzymes was based on
that of EID and MATTY (Aquaculture 1989, 79, 111-119). For this
purpose, the intestine of six one-year old rainbow trout
(Oncorhynchus mykiss) was exposed and processed as described in
example 22.
[0148] b) Procedure for the In Vitro Digestion Investigations
[0149] The in vitro investigations were carried out in analogy to
example 22 (see FIGS. 8 and 9).
TABLE-US-00002 TABLE 2 Sample Blank Precharge Substrate 0.143 mmol
0.143 mmol Met-Met (I) (40.1 mg) (40.1 mg) TRIS/HCl buffer
solution, 5.7 ml 9.8 ml pH 9.5 Reaction start Enzyme solution 4.2
ml -- ({circumflex over (=)} 10% trout solution) Reaction
37.degree. C. 37.degree. C. Reaction stop 0.2 ml of reaction
solution was taken up in 9.8 ml of 10% strength H.sub.3PO.sub.4
solution.
Example 24
In Vitro Digestion Experiments on DL-methionyl-DL-methionine (I)
with Digestive Enzymes From Omnivorous Shrimps
[0150] a) Isolation of the Digestive Enzymes from whiteleg shrimps
(Litopenaeus Vannamei)
[0151] The method for isolating the digestive enzymes was based on
that of Ezquerra and Garcia-Carreno (J. Food Biochem. 1999, 23,
59-74). For this purpose, the hepatopancreas was removed from five
kilograms of whiteleg shrimps (Litopenaeus Vannamei) and comminuted
together with crushed ice using a mixer. The further processing was
carried out in analogy to example 22.
[0152] b) Procedure for the In Vitro Digestion Investigations
[0153] The in vitro investigations were carried out in analogy to
example 22 (see FIGS. 10 and 11).
TABLE-US-00003 TABLE 3 Sample Blank Precharge Substrate 0.143 mmol
0.143 mmol Met-Met (I) (40.1 mg) (40.1 mg) TRIS/HCl buffer
solution, 5.7 ml 7.9 ml pH 9.5 Reaction start Enzyme solution 2.2
ml -- ({circumflex over (=)} 2 shrimps) Reaction 37.degree. C.
37.degree. C. Reaction stop 0.2 ml of reaction solution was taken
up in 9.8 ml of 10% strength H.sub.3PO.sub.4 solution.
Example 25
Biotransformation of D- to L-methionine with Enzymes From
Intestine, Liver and Pancreas of Common Carp
[0154] a) Isolation of the Digestive Enzymes from Common Carp
(Cyprinus carpio morpha noblis)
[0155] The method for isolating the digestive enzymes was based on
that of EID and MATTY (Aquaculture 1989, 79, 111-119). For this
purpose, the intestine of five one-year old common carp (Cyprinus
carpio morpha noblis) was exposed and processed as described in
example 22. To isolate liver enzymes, the livers were isolated,
homogenized and treated in analogy to the processing of the
intestinal enzymes in example 22. The procedure for enzyme
isolation from the pancreas was also analogous thereto.
[0156] b) Procedure for the In Vitro Biotransformation of D- to
L-methionine
[0157] D-Methionine was taken up in buffer solution, and the enzyme
solution was added. A blank without enzyme solution was made up in
each case as comparison and for estimating the purely chemical
transformation rate. After 24 hours, a sample was taken and the
composition was detected and quantified with the aid of calibrated
HPLC. The conversion was determined as the quotient of the area for
L-methionine and the area for D-methionine (see FIG. 4).
TABLE-US-00004 TABLE 4 Sample Blank Precharge Substrate 0.143 mmol
0.143 mmol D-Methionine (21.3 mg) (21.3 mg) Buffer solution 11.7 ml
23.4 ml Reaction start Enzyme cocktail 11.7 ml -- ({circumflex over
(=)} 5% carp solution) Reaction 37.degree. C. 37.degree. C.
Reaction stop 0.2 ml of reaction solution was taken up in 9.8 ml of
10% strength H.sub.3PO.sub.4 solution.
[0158] Buffer Solutions: Citrate buffer: pH 5, pH 6 and pH 7
Phosphate buffer: pH 8 TRIS/HCl buffer: pH 9
[0159] Enzyme cocktail composed of intestinal, hepatic and
pancreatic enzymes ({circumflex over (=)}5% carp solution): 2.6 ml
of enzyme solution from intestinal mucosa 3.5 ml of enzyme solution
from liver 5.6 ml of enzyme solution from pancreas
Example 26
Leaching Characteristics of the Mixtures of Methionylmethionine
Diastereomers LL/DD/LD/DL-I, DD/LL-I and DL/LD-I from Feed Pellets
Compared with DL-methionine, MHA and Calcium MHA
[0160] Feed Mixture:
[0161] The feed matrix used was a methionine-deficient feed mixture
of conventional ingredients such as, for example, soybean meal,
soybean oil, cornstarch, wheat meal, fish meal, cellulose,
crystalline essential amino acids and minerals and vitamins as
premixes. This mixture was then supplemented batchwise in 20 kg
batches in each case with the methionine derivatives stated in
table 5, with a 0.25% supplementation rate (based on sulfur
equivalents), and was homogenized and then pelleted with steam
treatment. As comparison with methionylmethionine (I), a pelleting
experiment was carried out in each case with DL-methionine, MHA
(methionine hydroxy analog) and calcium MHA. In addition, a control
experiment was carried out by pelleting without addition of a
methionine derivative (see table 5).
TABLE-US-00005 TABLE 5 Molecular Purity mass No. Methionine
derivative (wt %) (monomer) Initial weight 1 No additive -- -- 0.00
g 2 DL-Methionine 99.0% 149.21 50.61 g 3 MHA 88.0% 150.19 57.14 g 4
Calcium MHA (MHA-Ca) 93.3% 169.22 60.77 g 5 DD/LL/DL/LD methionyl-
99.7% 140.20 47.13 g methionine (I)
[0162] All the diastereomers of methionylmethionine (I) remained
stable throughout the pelleting process and steam treatment (see
table 6).
TABLE-US-00006 TABLE 6 Sample Feed mixture Feed pellets
supplemented supplemented Unsupplemented with with Parameter feed
mixture Met-Met (I) Met-Met (I) CP % 18.64 18.88 18.45 DM % 85.58
86.58 MET % 0.28 0.47 0.51 CYS % 0.32 0.32 0.30 MET + CYS % 0.59
0.79 0.81 LYS % 1.00 0.99 0.98 THR % 0.67 0.70 0.67 ARG % 1.16 1.19
1.17 ILE % 0.75 0.79 0.74 LEU % 1.54 1.60 1.51 VAL % 0.88 0.90 0.85
HIS % 0.47 0.51 0.48 PHE % 0.91 0.92 0.88 GLY % 0.78 0.81 0.77 SER
% 0.89 0.94 0.90 ALA % 0.89 0.93 0.89 ASP % 1.74 1.75 1.70 GLU %
3.62 3.79 3.58 MET-MET Ex 0.156 0.153 (I) MET Ex 0.017 0.022 LYS Ex
0.092 0.104 (Ex: soluble constituents)
[0163] In this case, the amino acid determination was based on EU
method 98/64/EC. After extraction of the free amino acids and
methionylmethionine (I), these were subsequently determined with
the aid of an amino acid analyzer by post-column derivatization
with ninhydrin (see table 6).
[0164] The leaching characteristics of the diastereomers of
methionylmethionine (I) from the feed pellets was then investigated
under water. In this case, the dissolving out of
methionylmethionine under water as a function of time, temperature,
water composition (salt or fresh water) was determined. For this
purpose, 20.0 g of the feed pellets were placed in a close-mesh
sieve bag and completely immersed in 200 g of water in an
Erlenmeyer flask. All the Erlenmeyer flask was subsequently
agitated continuously with a laboratory shaker at a constant
temperature of 20.degree. C. Then, at defined time intervals, a
sample of water was removed in each case and the content of the
individual pairs of methionylmethionine diastereomers in the water
was determined by HPLC (see table 7).
TABLE-US-00007 TABLE 7 DL/ LL/DD/ Time Methionine MHA MHA-Ca
LL/DD-I LD-I LD/DL-I 0 4.0% 6.0% 8.6% 2.7% 0.6% 1.5% 5 12.0% 12.8%
16.5% 3.7% 0.7% 2.0% 10 16.0% 20.8% 28.2% 6.5% 0.9% 3.2% 15 24.0%
28.8% 39.4% 7.7% 0.6% 3.6% 30 39.9% 50.5% 61.7% 12.1% 0.6% 5.4% 60
59.9% 75.4% 82.4% 20.6% 1.7% 9.5% 120 79.8% 94.1% 94.1% 27.4% 1.7%
12.3% 210 87.8% 99.9% 97.0% 35.9% 3.8% 17.0%
[0165] For comparison, in each case the feed pellets supplemented
with DL-methionine, MHA or calcium MHA were investigated under the
same conditions and thus their leaching characteristics under water
determined under the respective conditions (see FIG. 5 and table
7).
* * * * *