JPS63146851A - Production of myristylglycine and oligopeptide derivative thereof - Google Patents

Abstract

PURPOSE:To obtain the titled high-purity compound useful as a transformation inhibitor for cells, etc., by reacting myristic acid chloride with glycine, etc., in the presence of an alkali in an inert polar solvent or an aqueous solution containing the inert polar solvent. CONSTITUTION:Myristic acid chloride is reacted with glycine or NH2-end glycine oligopeptide in the presence of an alkali (e.g. NaHCO3, Na2CO3, NaOH, Na3PO4, etc.) in an inert polar solvent (e.g. scetonitrile, acetone, methyl ethyl ketone, etc.) or an aqueous solution containing the solvent to give myristylglycine or an oligopeptide derivative thereof. Existence of the polar solvent in the reaction system provides the aimed effective substance. The amount of the polar solvent added is preferably about 10-70wt%, more preferably 20-50wt% based on water.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing myristylglycine or a peptide derivative thereof.

It is generally known that the amino terminus of proteins is blocked by acetyl, pyroglutamyl, and formyl groups; found that myristic acid (a long-chain fatty acid) is covalently bound to the amino terminus of the catalytic subunit (Pro
c, Natl, Acad, Sci, MSA,
VOl, 79.8123-6131.1982),
The amino acid sequence of this myristyl terminal protein is
0 CHs-(CHa)+a-C-NH-Gly-Asn-
Ala-Ala-Ala-Ala--Lys-Lys- Since then, the presence of terminal myristyl groups has been successively reported in the following various proteins.

Calcineurin B: C) 13-(CH2)+2-C-NH-Gly-Asn
-Glu-Ala-(A, Aitkan et at
, FEBS LETTERS, Vol, 150°N
o, 2.314-318.1982) NADH-cytochrome b5 reductase: CHa-(CH2)+2-C-
NH-Gly-Ala-Gln-Leu-(J, Dz
ols et al, The Journal of
Biological Cheg+1try, Vo
l, 259. No, 21°13349-13354.
1984) Protein kinase p60src: ■ C) I3-(CH2)+2-C-NH-Gly-5er
-5er-Lys-5er--Lys-Pro-Lys
- (A, M, 5chultz et al, 5cie
nce, Vol, 227°427-429.198
5) In these reports, myristyl group, i.e. CHa-
The (CH2)+2-CO-NH- group is bonded to the amino-terminal glycine residue, as is clear from the above amino acid sequences, and the myristylation of the amino-terminal glycine residue is This suggests that it is crucial for certain mechanisms involved in transformation and growth control.

Furthermore, it is also disclosed that myristylglycine or myristylated oligopeptide can be obtained as a minimum unit by enzymatic digestion or chemical synthesis of the terminal myristylated protein. For example, AlM, 5
Chu l tz etc., the above p 60 src is NH2-
To identify it as a myristylated protein, this protein was digested with trypsin to My-Gly-
5er-5er-Lys, phylum y-c+y-Gln-Th
r and My-Gly (in the formula, My represents a myristyl group, the same applies hereinafter), and in order to identify these myristylated oligopeptides, synthetic My-Gly obtained from others was used.
Gly (synthesis method is unclear) and this synthesis My-
y-Gly-5 chemically synthesized from c+y by solid phase method
er-5er-Lys and My-Gly-Gln4h
r is used. In addition, for pressure cutting, etc., please refer to “Journal
of Chromatography, -356,1
79-185, (1986)'', My-c+y-c+y-'ryr-Arg,
My-Gly-Phe-NH2, My-Gly-NH2
and Myristyl oligopeptides such as My-c+y-c+y-Gly-N) 12 have been synthesized and identified.

However, research on the biological effects of myristyl groups in these proteins or oligopeptides on cells has just begun, and the specific effects or effects of myristyl glycine or myristyl groups have not yet been clarified. When obtaining glycine oligopeptides, conventional enzymatic digestion methods are generally obtained in the form of a mixture of myristylglycine and myristylglycine of various chain lengths, making separation and purification of the desired myristyl compound complicated. Conventional synthetic methods, such as using diimide,
Although the desired product was obtained, the yield was extremely low and was not practical.

The present inventors have developed a new and effective method for synthesizing myristylglycine or its peptide derivative (i.e., myristylglycine oligopeptide), which is thought to be useful for biological analysis of the 7-minoterminal myristyl group of proteins.
．． Furthermore, we have found that these synthesized myristylglycine or its oligopeptide derivatives have an inhibitory effect on the transformation of certain types of cells.

According to the present invention, myristic acid chloride and glycine or NH2-terminated glycine oligopeptide are reacted in the presence of an alkali in an inert polar solvent or an aqueous medium containing an inert polar solvent. A method for producing an oligopeptide derivative is provided. In addition, as a modification of the method for producing myristylglycine oligopeptide derivatives, myristylglycine is first synthesized by the above method, and then predetermined amino acid units are connected using a normal peptide synthesis method to produce a myristylglycine oligopeptide with the desired sequence. It can also be a peptide.

The above method of the present invention applies the so-called Schotten-Baumann reaction principle in which an amine is acylated with acid chloride in the presence of a dilute alkali. This is based on the fact that the target product could not be obtained substantially even if ordinary Schotten-Baumann reaction conditions were applied, and that an effective target product could only be obtained by the presence of a polar solvent as described below in the reaction system.

The myristic acid chloride used in the present invention can be prepared from commercially available myristic acid by a conventional method. Furthermore, commercially available glycine- or NHa-glycine-terminated oligopeptides, which are another starting material of the present invention, can be used, but the latter can also be obtained by adjusting the desired amino acid sequence using conventional peptide synthesis methods. Preferred examples of oligopeptides to be reacted with myristic acid chloride in the present invention are as follows.

NHa-Gly-Gly-COOII NH2-Gly-Ala-COOH NHa-Gly-Gly-Tyr-Arg-COOHN
Hz-Gly-Ala-Ala-Ala-Ala-CO
OHNHa-Gly-Phe-COOH NHa-Gly-5er-COOH The alkali used as a catalyst in the present invention may include any suitable alkali, such as NaHCO3, Na2COa, Na
OH, Na5pQ4, etc., and these have a molar concentration of about 0.1 to 1.0 molar concentration in the reaction solvent, preferably 0.3 to 0.0 molar concentration.
Used at 6 molar concentration.

In the present invention, the polar solvent which is an important factor for obtaining the target myristylglycine or myristylglycine oligopeptide includes nitrile solvents such as acetonitrile and propionitrile; ketones such as acetone, methyl ethyl ketone, and methyl isobutyl ketone; Can be used. However, although alcohols are the same polar solvents, they are not used in the present invention because they are active against myristic acid chloride. That is, the polar solvent used in the present invention must be inert to each starting reactant. Preferred polar solvents include nitrile solvents, particularly acetonitrile. Although the polar solvent used can be used as a reaction solvent in 100% form, it is preferably in the form of an aqueous solvent mixed with water, with a ratio of about 10% of the polar solvent to water.
It is used in the presence of ~70% by weight, preferably 20-50% by weight.

The reactions of the present invention can generally be carried out under stirring conditions at room temperature or below, and the reaction time is about 6 to 20 hours. After the reaction is complete, the resulting product is subjected to the usual methods such as acidic pH, extraction with ether, recrystallization with alcohol, etc.
A highly purified product can be obtained by subjecting it to a treatment.

The thus obtained myristylglycine or myristylglycine oligopeptide of the present invention is useful as a cell transformation inhibitor. Through experiments, the present inventors have found that the Rous sarcoma virus (RS), whose morphology changes significantly depending on temperature,
The effect of myristylglycine or myristylglycine oligopeptide synthesized according to the present invention on transformation was investigated using fibroblasts infected with the temperature mutant strain V tsNY68). That is, tsNY68rE%-stained chicken fibroblasts have the ability to transform into normal cells at 41'C, into cancer cells at 37°C, and return to normal cells again at 41'C.According to the present invention, It was found that addition of myristylglycine or myristylglycine oligopeptide to these transformed cells significantly inhibited transformation at 37°C. The amount added is infected cells (5 x 10')
10 to 50 μM is sufficient.

Hereinafter, the present invention will be specifically explained with reference to Examples.

Example 1 11.4g of glycine was dissolved in 150ml of 0.5M NaHCO3 aqueous solution containing 20% by weight of acetonitrile, 12.5g of myristyl chloride was slowly added dropwise with vigorous stirring, and the solution was heated to room temperature (with continued stirring). Approximately 2
The 0 reaction solution, which had been incubated overnight (around 0°C), was acidified with concentrated hydrochloric acid (p
H2,0) and the product was extracted with ether. The extract was then recrystallized with hot ethanol to obtain 8.2 g (71.9% yield) of white plate crystals. The melting point of this crystal is measured using a micro melting point meter (
Yanagimoto Seisakusho! The temperature was about 106-108°C when measured by In addition, the infrared absorption spectrum of this product measured by the KBr method has a wavelength of 1640 cm-1, as shown in Figure 1.
Absorption of an acid amide carbonyl group was observed in the vicinity, clearly showing the characteristics of myristylglycine (My-Gly-COOH).

Example 2 20g of Diglycine [Peptide Research Institute (Osaka)
] was dissolved in 150 ml of a 0.5 M NaHCO3 aqueous solution containing 30 wt% acetonitrile, and 12.5 g of myristyl chloride was slowly added dropwise while stirring vigorously. The apparatus was incubated overnight at 10°C. Hereinafter, in the same manner as in Example 1, 7.5 g of white needle-like crystals having a melting point of 185°C (yield 3
8%). The infrared absorption spectrum of this product by the KBr method clearly shows the characteristics of myristyldiglycine (My-Gly-Gly-COOH), as shown in FIG.

Example 3 In Example 2, instead of diglycine, glycine-alanine (NO3-Gly-Ala-COOH), glycine-glycine-tyrosine-arginine (NO3-Gly-
Gly-Ty+”ArH-C00H) and glycine-
The following three types of crystal products were obtained in the same manner as in Example 2 using serine (N) 12-Gly-5er-COOH). All oligopeptides used were obtained as commercial products from Peptide Research Institute (Osaka).

LL! ! i Ship My-Gl
y-Ala-COOH119℃My-Gly-Gly-
Tyr-Ar8-COOH144℃My-Gly-5e
r-COOH 116°C Example 4 10 day old embryonated chicken egg fibroblasts were prepared by a conventional method. The prepared cells were first cultured at 41°C for 48 hours, then incubated at 37°C.
After culturing at ℃ for 48 hours, the temperature was returned to 41'C and culture was continued for an additional 48 hours. When observing the morphological changes of cells at each culture stage using an inverted microscope, the 3rd a.
- As shown in figure c (vs. 1), in normal cells, cell division and proliferation are observed in all three culture stages, as well as directionality and anchorage dependence (anchorage pendence). ) have been well observed. Also, RSV (AsNY60 stock...
... Cells (5x 10
Cells infected at a volume of 1 ml per cell were cultured in three stages in exactly the same manner as described above, and the results shown in Figures 4a-c were obtained when observed using an inverted microscope (Control 2). As is clear from these results, in both stages (Figs. 4a and 4c), the same directionality as in Figs. 3a and 3c is observed in normal cells, whereas in 41"C 148-hour culture, 37 When cultured at °C, cell division and proliferation were rapid, directionality was lost, and transformation was clearly occurring.

Furthermore, myristylglycine prepared in Example 1 was added to cells infected with AsNY68 strain of control 2 (5x
50 gM per 10' pieces) was added at 48°C.
When the phenotypic changes were observed after culturing at 37° C., the results shown in FIGS. 5a and 5b were obtained, respectively. Culture at 37℃! In I (Figure 5b), substantially the same phenotypic state as in the 48°C culture (Figure 5a) was observed, with sufficient directionality and anchorage dependence being observed, and cell division and proliferation being suppressed. I understand that.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum diagram of myristylglycine prepared in Example 1. FIG. 2 is an infrared absorption spectrum diagram of myristyl diglycine prepared in Example 2. Figure 3a--C shows 41 cells of normally developed chicken egg fibroblasts.
These are inverted micrographs (magnification = 150 times) showing morphological changes in cells under each culture condition of "C (first stage), 37"C and 41"C (second stage). Figures 4a-c show R3V-infected embryonated chicken egg blast cells at 41°C (
These are inverted micrographs (magnification: 150x) showing morphological changes in cells under culture conditions of (first part), 37°C, and 41'C (second part). Figures 5a-b are inverted micrographs showing the morphological changes of R5V-infected cells cultured at 41'C and 37C when myristylglycine of the present invention was added (magnification: 150x). ■ 1 Figure 41 "C1413R1'': Il
37℃, 48 hours Fig. 3c 41℃, 4E1M leap

Claims (3)

[Claims] (1) Myristic acid chloride and glycine or NH_2
- A method for producing myristylglycine or an oligopeptide derivative thereof, which comprises reacting a terminal glycine oligopeptide in an inert polar solvent or an aqueous solvent containing an inert polar solvent in the presence of an alkali. (2) Myristylglycine is obtained by reacting myristic acid chloride and glycine in the presence of an alkali in an inert polar solvent or an aqueous solvent containing an inert polar solvent, and this myristylglycine is converted into an amino acid or A method for producing a myristylglycine oligopeptide derivative, which comprises obtaining a desired myristylglycine oligopeptide derivative by reacting with an oligopeptide. (3) A cell transformation or growth inhibitor consisting of myristylglycine or an oligopeptide derivative thereof.