Glycopeptides and method for preparing same

Glycopeptides according to the present invention have the formula: ##STR1## wherein n=1 or 2; R is a residue of an aminoacid or linear peptides built of 2 to 5 amino acid residues. These compounds are prepared by the method involving condensation of unblocked muramyl-containing N-acetylamino-sugars of the formula: ##STR2## wherein n=1 or 2, with blocked aminoacids or peptides. The method of this invention makes it possible to produce synthetic glycopeptides manifesting a biological activity. These compounds possess an antitumor and adjuvant activity.

The present invention relates to the art of biologically active compounds 
and, more specifically, to glycopeptides and a method for preparing same. 
FIELD OF THE INVENTION 
Glycopeptides constitute a broad class of organic compounds comprising 
substances including the sugar part and the peptide part. Cell walls of 
bacteria are constituted by glycopeptides. The sugar part of glycopeptides 
incorporates an unusual carboxyl-containing 
aminosugar-2-acetamido-3-O-/D-1'-carboxyethyl/-2-deoxy-.beta.-D-glucopyran 
ose generally referred to as N-acetylmuramic acid (MurNAc) and found only 
in cell walls of bacteria. 
Glycopeptides of bacterial cell walls comprise gigantic polymeric molecules 
composed of alternating units of a disaccharide 
GlcNAc/1.fwdarw.4/MurNAc[N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramic 
acid] with peptides of a similar structure bonded with muramic acid 
(so-called basic peptide chains). Rigidity of the cell wall structure of 
bacteria is due to peptide bonds (or peptide bridges) between basic 
peptide chains. By means of a specific enzymatic hydrolysis it is possible 
to break-up certain bonds in a polymeric molecule of glycopeptides of cell 
walls of bacteria and obtain glycopeptide fragments of a various length 
and various structure /1. Ghuysen J.-M., Bact. Rev. 32, No. 4, 425 (1968), 
2. Schliefer K. H., Kandler O., Bact. Rev. 36, No. 4, 407, (1972)/. 
Until recently it has been believed that the role of glycopeptides of cell 
walls of bacteria resides merely in providing rigidity of the cell walls 
for protection of the bacterial cell from the effect of the ambient 
medium. However, with the development of investigations on adjuvant 
properties of mycobacterial cells (preparations BCG, Freund's complete 
adjuvant) it has been found that numerous glycopeptides recovered from 
bacterial cell walls are strong adjuvants (cf. Ellous F., Adam A., 
Ciorbaru R., Lederer E., Bioch. Bioph. Res. Comm. 59, No. 4, 1317 (1974)). 
(Adjuvants are compounds causing non-specific stimulation of the immune 
system of a human being and animals which results in an increased 
production of antibodies and in an enhancement of protective reaction of 
the organism, against e.g. infection, is increased. Adjuvants are used in 
medicine for the manufacture of vaccines and sera). 
BACKGROUND OF THE INVENTION 
Known in the art (Kusumoto S., Tarumi Y., Ikenaka K., Shiba T., Bull. Chem. 
Soc. (Japan) 49, (2), 533-539 (1976); and Lefrancier P., Choay J., Derrien 
M., Lederman I., Int. J. Pept. Protein Res., 9, No. 4, 249-257 (1977) is 
synthesis of certain fragments and analogues of glycopeptides of cell 
walls of bacteria, namely N-acetylmuramylpeptides of the formula: 
##STR3## 
wherein R is residues of aminoacids or peptides such as Ala, Gly, 
Ala-D-Glu, Ala-D-Glu-NH.sub.2, 
##STR4## 
D-Ala-D-Glu-NH.sub.2 and the like. 
The synthesis comprises condensation of benzyl esters of corresponding 
aminoacids or peptides with blocked N-acetylmuramic acid. 
This prior art method for the preparation of said glycopeptides has a 
disadvantage residing in that into the condensation reaction only blocked 
N-acetylmuramic acid 
(benzyl-2-acetamido-4,6-benzylidene-3-O-/D-1'-carboxyethyl/-2-deoxy-.alpha 
.-D-glucopyranoside/ is introduced which is preliminary synthetized in 
three stages from N-acetylglucosamine with a yield of 17-22% following the 
below-given scheme: 
##STR5## 
Some of the above-mentioned synthetic N-acetylmuramylpeptides are strong 
adjuvants. 
In a joint paper by Soviet and Bulgarian scientists Kotani S., Watanabe Y., 
Kinoshita F., Shimono T., Morisaki I., Shiba T., Kusumto S., Tarumi Y., 
Ikenaka K., Biken J. 18, 105 (1975)) it has been shown that a mixture of 
glycopeptides isolated from cell walls of Lactobacillus bulgarieus has an 
unusual antitumor activity: neutral glycopeptides of Lactobacillus 
bulgarieus cause necrosis of the tumor tissue on mice with sarcoma S-180 
(Bogdanov I. G., Dalev P. G., Gurevich A. I., Kolosov M. N., Malkova V. 
P., Plemyannikova L. A., Sorokina I. B., Febs. Letters 57, No. 3, 259 
(1975)). The synthesis of these glycopeptides is hitherto unknown. 
The detection of biological activity (adjuvant activity and ability to 
cause necrosis in a tumor tissue) of glycopeptides isolated from cell 
walls of bacteria has given impetus to a synthetic preparation of various 
fragments of naturally-occurring glycopeptides and analogues thereof 
necessary for a profound investigation of biological activity of these 
compounds. 
Analysis of fragments of glycopeptides isolated from cell walls of 
different microorganisms and demonstrating adjuvant activity, as well as 
analysis of glycopeptides of Lactobacillus bulgarieus revealing an unusual 
antitumor activity has shown that both N-acetylmuramic acid and 
N-acetyl-glucosamine are incorporated in the sugar part of the molecules 
of glycopeptides. However, the synthesis of glycopeptides containing, 
apart from N-acetylmuramic acid, N-acetylglucosamine, i.e. the synthesis 
of N-acetylglucosaminyl-N-acetylmuramylpeptides has not been hitherto 
known from the literature. The prior art method for the synthesis of said 
muramylpeptides makes it possible to obtain only N-acetylmuramylpeptides. 
The multi-stage synthesis scheme employed for this purpose is unsuitable 
in principle for association of the residue of N-acetylglucosamine with 
N-acetylmuramic acid by means of the bond 1.fwdarw.4 (all residues of 
aminosugars in any glycopeptide of cell walls of bacteria are 
interconnected by the bond 1.fwdarw.4). Therefore, the known method for 
preparing N-acetylmuramylpeptides cannot be used for the synthesis of 
N-acetylglucosaminyl-/1.fwdarw.4/-N-acetyl muramylpeptides. This defined 
the attempts in finding a new method for the preparation of various 
fragments and analogues of naturally-occurring glycopeptides of cell walls 
of bacteria. 
OBJECT OF THE INVENTION 
It is an object of the present invention to provide novel biologically 
active synthetic glycopeptides incorporating in the sugar part of their 
molecules various fragments of aminosugars of the type normally found in 
the cell walls of bacteria and to develop also a novel method for the 
preparation of said glycopeptides. 
SUMMARY OF THE INVENTION 
The object of the present invention is accomplished by glycopeptides of the 
formula (I) hereinbelow, wherein n=1 or 2, R is a residue of an aminoacid 
or linear peptides built of 2 to 5 amino acid residues. 
##STR6## 
Glycopeptides of this formula are novel chemical compounds. It has been 
found that said compounds are biologically active and useful in medicine. 
The synthesis and biological activity of the glycopeptides according to 
the present invention have not been hitherto described in the literature. 
The object of the present invention is accomplished also by a novel method 
of preparing glycopeptides of formula (I) which comprises condensation of 
unblocked (unprotected) muramyl-containing N-acetylaminosugars of the 
formula: 
##STR7## 
wherein n=1, 2 with blocked linear peptides by way of activation of the 
carboxy group of said aminosugars. 
The method according to the present invention provides broad opportunities 
for the preparation of a number of naturally-occurring glycopeptides and 
analogues thereof by way of synthesis. 
Other objects and advantages of the present invention will now become more 
fully apparent from the following detailed description. 
DETAILED DESCRIPTION OF THE INVENTION 
As it has been already mentioned hereinbefore, the method according to the 
present invention for the synthesis of glycopeptides of the general 
formula (I) comprises condensation of the sugar component with blocked 
linear peptides. Said method is characterized by unblocked 
muramyl-containing N-acetylaminosugars of formula (II) being introduced 
into the reaction of condensation as the sugar component. 
The condensation reaction is conducted in an inert solvent medium, 
preferably in the presence of a condensation agent such as Woodward 
reagent (N-ethyl-5-phenyl-isoxazolium-3'-sulphonate) at a temperature 
within the range of from 0.degree. to 25.degree. C. in one stage. 
The subsequent removal of the blocking groups from the aminoacid or peptide 
residue is effected by conventional techniques. 
The starting aminosugars of formula (II) are isolated from the biomass of 
Micrococcus lysodeicticus, since the synthesis of such muramyl-containing 
N-acetylaminosugars is not known. 
The employed method for isolating aminosugars of formula (II) from the 
biomass is known and described in the literature. It involves enzymatic 
hydrolysis of the biomass of Micrococcus lysodeikticus by means of 
lysozyme and a further two-stage purification in columns packed with Dowex 
50.times.8 (H.sup.+ -form) with particles of 200-400 mesh and Dowex 
1.times.8 (CH.sub.3 COO.sup.- -form) 200-400 mesh (Hoshino O., Zenavi U., 
Sinay P., Jeanloz R. W., J. Biol. Chem. 247, No. 2, 381 (1972); and Sharon 
N., Osawa T., Flowers H. M., Jeanloz R. W., J. Biol. Chemistry, 241, 223 
(1966)). 
Analysis data for aminosugars of formula (II): GleNAc-/1.fwdarw.4/MurNAc: 
aminoacid and aminosugar analysis /6 N HCl, 2 and 24 hours, 100.degree. 
C./Mur:GlcNH.sub.2 1.0:1.0 /2 hours/, 1.00:1.03 /24 hours with the account 
of decomposition/, no aminoacids; 
EQU [.alpha.].sub.D.sup.18.degree. +16.8.degree..fwdarw.+12.0.degree./c 
0.5,H.sub.2 O/ 
EQU [.alpha.].sub.D.sup.16.degree. +15.2.fwdarw.+10.2.degree./c0.5,H.sub.2 o,24 
hours/, 
Found, %: C,43.88; H,6.70; N,5.00. Calculated for C.sub.19 H.sub.32 N.sub.2 
O.sub.13.1.5 H.sub.2 O: C,43.59; H,6.74; N,5.30. 
GlcNAc/1.fwdarw.4/MurNAc/1.fwdarw.4/GlcNAc/1.fwdarw.4/MurNAc 
aminoacid and aminosugar analysis: /6 N HCl, 100.degree. C., 16 and 24 
hours/, Mur:GlcNH.sub.2 1.0:1.0 /with the account of decomposition at the 
16-th hour/ and 1.00:1.04 (with the account of decomposition at 24 hours/, 
no aminoacids; 
EQU [.alpha.].sub.D.sup.18.degree. 
-10.4.degree..fwdarw.-13.1.degree./c0.5,H.sub.2 O,24 hours/ 
Found, %: C,44.50; H,5.63; N, 5.00. Calculated for C.sub.38 H.sub.62 
N.sub.4 O.sub.25.3H.sub.2 O, %: C,44.35; H,6.60; N, 5.45. 
The starting blocked aminoacids or peptides employed for the synthesis are 
obtained by known methods of the peptide chemistry. In doing so, as a 
rule, use is made of benzyl ethers for blocking C-terminal carboxy groups 
and tert. butyloxycarbonyl and benzyloxycarbonyl groups for protection of 
free amino groups. 
According to the present invention there are prepared glycopeptides of the 
general formula (I) including: 
I. GlcNAc/1.fwdarw.4/MurNAc-Ala 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanine 
II. GlcNAc/1.fwdarw.4/MurNAc-Ala-D-Glu-NH.sub.2 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-isoglutamine 
III. GlcNAc/1.fwdarw.4/MurNAc-Ala-D-Glu 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-glutamic acid 
IV. 
##STR8## 
amide of N.sup..epsilon. 
-[N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-glutamyl-D-iso- 
asparaginyl]-lysyl-D-alanine 
V. 
##STR9## 
/1.fwdarw.4/-bis-[N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D 
- 
isoglutamine] 
The above-listed compounds comprise lyophilically dried white hygroscopic 
powders soluble in water, physiological solution, methanol or 50% ethanol, 
but insoluble in ether, benzene, petroleum ether or chloroform. These 
compounds possess a biological activity. As to the nature of this 
biological activity, these glycopeptides principally differ from antitumor 
preparations employed currently in oncological practice. The currently 
employed preparations, as a rule, cause merely retardation of the tumor 
growth and this effect is most pronounced at the early stages of the 
malignant tumor growth, i.e. the known currently employed preparations are 
effective in the treatment of early stages of the disease. All of them are 
substantially cytotoxic in respect of cells of normal tissues as well, 
they frequently show immunodepressant properties, inhibit hemopoiesis 
(hematoplasty). In contrast thereto, the biological activity of the 
synthetic glycopeptides according to the present invention is revealed in 
a rapid selective necrosis of a strongly developed tumor and a subsequent 
retardation of the tumor growth even after a single injection of the 
compound. Normal tissues are not injured therewith. All the glycopeptides 
according to the present invention are of low toxicity and cause no 
inhibition of hemopoiesis. 
Biological tests have been performed on mice with a 8-day Krocker sarcoma 
(S-180): intravenous administration (dose of 50 to 100 mg/kg/; necrosis 
evaluation after 24 hours (4-6 as of the 6-point scale), measurement of 
volume (or weight) of the tumor after 4-5 days (growth inhibition of up to 
70%). For example, upon a single intravenous administration of 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-iso-glutamine 
(Compound II) to mice with a 8-day sarcoma S-180 (strongly developed 
tumor) in the dose of 100 mg/kg with four out of five test animals a 
selective necrosis of the tumor tissue covering substantially the entire 
tumor is observed 24 hours after administration, while after 5 days 70% 
inhibition of the growth of the malignant tumor occurs as compared to the 
control animals. Therewith, no signs of toxicity are noticed, hemopoiesis 
is not inhibited, the number of leukocytes is substantially increased. 
We have demonstrated that the necrosis is not caused by the constituent 
components of said glycopeptides: N-acetylglucosamine (VI), 
N-acetylmuramic acid (VII), 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramic acid (VIII), 
N-acetylglucosaminyl 
/1.fwdarw.4/-N-acetylmuramyl/1.fwdarw.4/N-acetylglucosaminyl/1.fwdarw.4/N- 
acetylmuramic acid (IX) or by peptides employed for the synthesis of 
compounds I to V: Ala-D-Glu-NH.sub.2 (X), Ala-D-Glu (XI),

All these data are shown in the Table following the Examples. 
For a better understanding of the present invention some specific Examples 
illustrating preparation of glycopeptides are given hereinbelow. 
Melting temperatures are determined in the Koffler block (temperatures are 
corrected). Aminoacid and aminosugar analyses are carried out using an 
automatic aminoacid analyser Liquimat (produced by Labotron company). 
Rotation angles are determined on Perkin-Elmer polarimeter 141. The 
systems for thin-layer chromatography (TLC): n-BuOH:EtOH:H.sub.2 O 3:6:1 
(A); n-BuOH:AcOH:H.sub.2 O 3:1:1 (B), n-BuOH:AcOH:H.sub.2 O 2:1:1 (C). 
EXAMPLE 1 
N-acetyl-glucosaminyl/1.fwdarw.4/N-acetylmuramylalanine (I) 
(a) To a solution of 160 mg (0.33 mM) of 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramic acid in 8 ml of 
dimethylformamide (DMFA) at the temperature of 0.degree. C. there are 
added 0.05 ml of triethylamine (TEA) and 150 mg (0.45 mM) of Woodward 
reagent K; the mixture is stirred for 1 hour at 0.degree. C. and then for 
one more hour at the temperature of 20.degree. C. till complete 
dissolution. A solution of 90 mg (0.3 mM) of nitrobenzyl ether of alanine 
bromohydrate and 0.05 ml of triethylamine (TEA) in 4 ml of DMFA are 
dropwise added under stirring for 15 minutes and stirring is continued for 
20 hours at room temperature. The residue after evaporation in vacuum is 
dissolved in 1 ml of a 50% aqueous ethanol and passed through a column /10 
ml/ with packing of Dowex 50.times.8 (H.sup.+ -form) and then through a 
column /10 ml/ packed with Dowex 1.times.8 (CH.sub.3 COO.sup.- -form), in 
both cases elution being effected by means of a 50% aqueous ethanol. The 
residue after evaporation of the eluate is resettled from methanol by 
means of ether. There are produced 80 mg (33.0%) of nitrobenzyl ether of 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramylalanine. 50 mg of the 
latter are dissolved in a 75% aqueous acetic acid and hydrogenated over 
Pd-black for 6 hours. The catalyst is filtered-off and the residue after 
evaporation is resettled from methanol by ether to give 30 mg /80%/ of the 
product (I). Found, %: C,44.41; H,6.78; N,7.06; Calculated for C.sub.22 
H.sub.37 N.sub.3 O.sub.4.1.5H.sub.2 O,%: C, 44.44; H,6.78; N,7.07. 
Aminoacid and aminosugar analyses: /A.A.A./ (6 N HCl, 100.degree. C., 16 
hours): Ala:Mur:GlcNH.sub.2 1:1.08:1.1 (with the account of decomposition 
at the 16-th hour of hydrolysis); [.alpha.].sub.D.sup.18.degree. 
-3.5.degree.(c 0.5, H.sub.2 O) M.p. 150.degree. C. (with decomposition). 
Thin-layer chromatography (TLC): R.sub.f =0.45. 
System A 
(b) To a solution of 0.16 g (0.33 mM) of 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramic acid and 0.045 ml of TEA 
in 10 ml of DMFA under stirring at -15.degree. C. there is added 0.045 ml 
(0.33 mM) of isobutylchloroformiate. Two minutes after a solution of 0.07 
g (0.22 mM) of bromohydrate of nitrobenzyl ether of alanine cooled to 
-15.degree. C. and 0.036 ml of TEA in 10 ml of DMFA are added thereto. The 
solution is maintained for three hours at the temperature of -15.degree. 
C. under stirring. The residue after evaporation in vacuum is subjected to 
a further purification following the procedure of the foregoing Example 
1(a) to give 24 mg (32%) of nitrobenzyl ether of 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramylalanine. After the removal 
of the blocking group as described in Example 1(a) hereinabove, there are 
obtained 17 mg /80%/ of the product (I). 
EXAMPLE 2 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-isoglutamine (II) 
The reaction of condensation is conducted as described in the foregoing 
Example 1(a) starting from 130 mg (0.3 mM) of benzyl ether of 
alanyl-D-isoglutamine trifluoroacetate. The reaction mixture is evaporated 
to a small (about 1 ml) volume; the precipitate settled-out by means of 
n-butanol is filtered-off and dissolved in 1 ml of a 50% aqueous ethanol; 
further purification is conducted following the procedure of Example 1 to 
give 75 mg (31%) of benzyl ether of N-acetyl 
glucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-isoglutamine. 
Hydrogenation over Pd-black is effected as in Example 1 with 50 mg of said 
benzyl ether. There are obtained 36 mg (81%) of the product (II). Found, 
%: C,39.64; H,7.11; N,8.41; calculated for C.sub.27 H.sub.45 N.sub.5 
O.sub.16.7H.sub.2 O, %: C,39.22; H,7.23; N,8.51. M.W. 695, 697; A.A.A.: (6 
N HCl, 100.degree., 16 hours): Ala-Glu:Mur:GlcNH.sub.2 1:0.81:1.01:1.02 (6 
N HCl, 100.degree., 24 hours) 1:0.96:0.96:1.2 (with the account of 
decomposition at 16 and 24 hours respectively); 
[.alpha.].sub.D.sup.20.degree. +2.8.degree. (c with 0.5, H.sub.2 O, after 
5 minutes), 0.degree. C. (c 0.5, H.sub.2 O, after 22 hours), M.p. 
170.degree. C. with decomposition; TLC: R.sub.f =0.3 (System A), R.sub.f 
=0.45 (System C). 
EXAMPLE 3 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-glutamic acid 
(III) 
The reaction of condensation and treatment of the reaction mixture are 
conducted following the procedure described in the foregoing Example 1a, 
with 310 mg (0.6 mM) of dibenzyl ether of alanyl-D-glutamic acid 
trifluoroacetate. There are obtained 200 mg (38%) of dibenzyl ether of 
N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-glutamic acid; 
after the removal of benzyl blocking groups by hydrogenolysis over 
Pd-black from 100 mg of said dibenzyl ether there are obtained 64 mg (80%) 
of the product (III). Found, %: C,43.00; H,6.71; N,7.30. Calculated for 
C.sub.27 H.sub.44 N.sub.4 O.sub.17.3H.sub.2 O, %: C,43.19; H,6.71; N,7.46. 
M.W. (molecular weight): 696, 681. A.A.A. (6 N HCl, 100.degree. C., 24 
hours): Ala.div.Glu:Mur:GlcNH.sub.2 1.04:1.00:0.96:1.00 (with the account 
of decomposition at the 24-th hour). [.alpha.].sub.D.sup.18.degree. 
-4.2.degree. (with 0.5 H.sub.2 O), M.p. 170.degree. C. (decomposition), 
TLC: R.sub.f =0.2 (System A). 
EXAMPLE 4 
Amide of N.sup..epsilon. 
-[N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-glutamyl-D-iso- 
asparaginyl]-lysyl-D-alanine (IV) 
The condensation reaction is conducted as described in Example 1(a) 
hereinbefore with 300 mg (0.34 mM) of amide of N.sup..alpha. -carbobenzoxy 
N.sup..epsilon. 
[alanyl-(.gamma.-benzyl)-D-glutamyl-D-isoasparaginyl]-lysyl-D-alanine 
trifluoroacetate. The reaction mixture is evaporated to the volume of 
about 1 ml, the condensation product is precipitated with water, washed 
with water, methanol and ether to give 136 mg (27%) of amide of 
N.sup..alpha. -carbobenzoxy-N.sup..epsilon. 
-[N-acetylglucosaminyl/1.fwdarw.4/N-acetomuramyl-alanyl-(.alpha.-benzyl)-D 
-glutamyl-D-iso-asparaginyl]-lysyl-D-alanine and, after the removal of 
benzyloxycarbonyl and benzyl protective groups by hydrogenolysis over 
Pd-black, from 50 mg there are produced 35 mg (83%) of the product (IV). 
Found, %: C,52.32; H,6.55; N,11.00; calculated for C.sub.55 H.sub.80 
N.sub.10 O.sub.22.1.5H.sub.2 O, %: C,52.41; H,6.63; N,11.11. M.W. 
1,233.32; A.A.A. (6 N HCl, 100.degree. C., 16 hours): 
Asp:Lys:Glu:Ala:Mur:GlcNH.sub.2 1.00:0.96:1.04:2.20:1.00:0.80 (with the 
account of decomposition at the 16-th hour). TLC: R.sub.f =0.3 (System B). 
EXAMPLE 5 
1.fwdarw.4/bis-[N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramyl-alanyl-D-is 
oglutamine] (V) 
The reaction of condensation is conducted following the procedure described 
in Example 1(a) hereinbefore with 243 mg (0.25 mM) of 
N-acetylglucosaminyl/1.fwdarw.4/-Nacetylmuramyl/1.fwdarw.4N-acetylglucosam 
inyl/1.fwdarw.4/N-acetylmuramic acid and 210 mg (0.5 mM) of benzyl either 
of alanyl-D-isoglutamine trifluoroacetate. The reaction mixture is 
evaporated to the volume of about one ml, added with 80% of water, 
filtered and passed successively through columns filled with Dowex 
50.times.8 (H.sup.+ -form) and Dowex 1.times.8 (CH.sub.3 COO.sup.- -form). 
The resulting 50 mg of crude condensation product are divided in 
preparative plates "Merck" in the system n-BuOH:EtOH:H.sub.2 O 3:6:1 to 
give 18 mg of 
/1.fwdarw.4/-bis[N-acetylglucosaminyl/1.fwdarw.4/N-acetylmuramylalanyl-(.a 
lpha.-benzyl)-D-isoglutamine] and, after the removal of benzyl protective 
groups there are obtained 10 mg of the product (V). Found, %: C,44.01; 
H,6.74; N,9.44; Calculated for C.sub.53 H.sub.86 N.sub.10 
O.sub.29.6H.sub.2 O %: C,44.34; H,6.88; N, 9.76; M.W. 1,327; 351; A.A.A. 
(6 N HCl, 100.degree. C., 16 hours), Ala.div.Glu:Mur:GlcNH.sub.2 
1.00:1.01:1.00:1.05; TLC: R.sub.f =0.1 (System C). 
TABLE 
__________________________________________________________________________ 
Necrotic 
reaction 
Inhibition 
shown aft- 
of the tu- 
Death 
ter 24 h, 
mor growth, 
of 
Dose, 
number of 
% of the 
ani- 
Compound mg/kg 
animals 
control 
mals 
__________________________________________________________________________ 
I. N--acetylglucosaminyl/1.fwdarw.4/N--acetyl- 
100 3/5 19 0 
muramyl-alanine 50 2/5 17 0 
II. N--acetylglucosaminyl/1.fwdarw.4/N--acetyl- 
100 4/5 70 0 
muramylalanyl-D-iso-glutamine 
50 8/10 12 0 
III. 
N--acetylglucosaminyl/1.fwdarw.4/N--acetyl- 
100 3/5 45 0 
muramulalanyl-D-glutamic acid 
50 4/10 +28 0 
IV. Amide of N.sup..epsilon. [N--acetylglucosaminyl/1- 
100 3/5 41 0 
.fwdarw.4/N--acetylmuramylalanyl-D-glutamyl-D- 
50 3/5 23 0 
iso-asparaginyl]-lysyl-D-alanine 
V. /1.fwdarw.4/-bis-[N--acetylglucosaminyl/1.fwdarw.4/ 
50 3/5 66 0 
N--acetylmuramylalanyl-D-isoglutamine] 
Constituents of said compounds 
VI. N--acetylglucosamine 250 0.5 0 0 
VII. 
N--acetylmuramic acid 250 0/8 52 25 
100 0/8 37 0 
VIII. 
N--acetylglucosaminyl/.fwdarw.4/ 
250 0/10 0 0 
N--acetylmuramic acid 100 0/10 0 0 
IX. N--acetylglucosaminyl/1.fwdarw.4/N--acetyl- 
250 0/10 0 0 
muramyl/1.fwdarw.4/N--acetylglucosaminyl- 
/1.fwdarw.4/N--acetylmuramic acid 
X. Alanyl-D-iso-glutamine 
100 0/10 +28 0 
XI. Alanyl-D-glutamic acid 
100 0/8 37.3 0 
XII. 
Amide of N.sup..epsilon. [Alanyl-D-glutamyl-D-iso- 
250 0/5 0 0 
asparaginyl]-lysyl-D-alanine 
__________________________________________________________________________