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  Pharmaceutical Patents  

 

Title:  Method of producing partial peptide of enolase protein from Plasmodium falciparum
United States Patent: 
7,713,926
Issued: 
May 11, 2010

Inventors: 
Oku; Hiroyuki (Maebashi, JP), Omi; Kazuto (Maebashi, JP), Kuriyama; Keisuke (Sendal, JP), Yamamoto; Jyunya (Kiryu, JP), Yamada; Keiichi (Kiryu, JP), Katakai; Ryoichi (Kiryu, JP), Sato; Kumiko (Yoshioka-machi, JP), Suzuki; Mamoru (Takasaki, JP), Kawazu; Shin-ichiro (Tokyo, JP), Kano; Shigeyuki (Tokyo, JP)
Assignee: 
National University Corporation Gunma University (Maebashi-shi, Gunma, JP)
Appl. No.: 
11/663,962
Filed: 
September 28, 2005
PCT Filed: 
September 28, 2005
PCT No.: 
PCT/JP2005/017851
371(c)(1),(2),(4) Date: 
March 28, 2007
PCT Pub. No.: 
WO2006/035815
PCT Pub. Date: 
April 06, 2006

 

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Abstract

The peptide production method of the present invention produces a peptide (SEQ ID NO: 1) of a protein from Plasmodium falciparum, which is effective as a malaria vaccine. The method produces the peptide of SEQ ID NO: 1 by linking the fragments (i) through (v) shown below: (v) Asn-Asn-Asp-Xaa (SEQ ID NO: 2); (iv) Asp-Phe-Lys-Thr-Pro (SEQ ID NO: 3); (iii) Asn-Lys-Thr-Tyr-Asp-Leu (SEQ ID NO: 4); (ii) Phe-Tyr-Asn-Ser-Glu (SEQ ID NO: 5); and (i) Xaa-Ala-Ser-Glu (SEQ ID NO: 6), where `Xaa` in (i) and (v) represents zero or any arbitrary number of amino acid residues.

Description of the Invention

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. .sctn.371 to International Application No. PCT/JP2005/017851, filed Sep. 28, 2005, which claims priority Japanese Patent Application No. 2004-281518, filed Sep. 28, 2004, both of which applications are expressly incorporated herein by reference in their entirety.

1. TECHNICAL FIELD

The present invention relates to a method of producing a partial peptide of enolase protein from Plasmodium falciparum. The present invention also relates to a method of producing medicines including a peptide with an ability of inducing an immunological response to Plasmodium falciparum by taking advantage of an immunological reaction in human or another animal, a diagnostic agent of the immunological state of malaria infection, and an immunological antigen peptide of inhibiting the proliferation of Plasmodium falciparum.

2. BACKGROUND ART

(1) Present Status of Malaria and Other Infections

In spite of the optimistic prospective of overcoming various infections, the global transportation of people and products, the development-inducing global environmental change, and the drastic change of various social activities have significantly and rather adversely changed the circumstances surrounding the infections. For example, the popularity of overseas travels and the destruction of rainforests induce tropical diseases, and the heavy use and the abuse of anti-infectives lead to the appearance of drug-resistant viruses and bacteria.

Among the variety of infections, especially the actions against malaria as a tropical protozoal infection are slow and insufficient. The number of malaria patients amounts to approximately 300 to 500 millions per year including 1.5 to 2.7 million deaths (see WHO report, 1999). Among four human-infecting protozoa species, Plasmodium falciparum is most severe and deadly. Malaria has the massive impact on the human health and also causes the economic recession and social instability in African states.

It is often pointed out that the destruction of rainforests and the global warming have contribution to the increasing number of malaria patients. According to the reports of the International Panel on Climate Change (1996 and 1998), a potential increase of 50 to 80 million new malaria patients would be predicted by a temperature rise of 2.degree. C. by the global warming. With the wide spread of overseas travels, the number of malaria-(imported malaria-) infected individuals in Japan has an increasing tendency to 120 to 150 patients per year from 50 to 70 patients per year in 1980s.

(2) Prior Arts and Their Problems

Many of currently used malaria medications include the patent loyalties and are thus rather expensive. This interferes with the widespread of the malaria medication in the developing countries. There are some inexpensive malaria drugs like chloroquine. The limitless use of these popular drugs leads to the high degree of drug resistance.

There are many malaria-related issues to be solved. The domestic and international pharmaceutical industries have not actively been involved in development of therapeutic and preventive medicines for malaria. The target of their research and development is focused on the age-related disorders and diseases, which have the greater importance for the developed countries. Another reason for their sluggish attitude is a relatively small market of the products for the developing countries. The existing companies may thus not be sufficiently reliable for development of novel antimalarial pharmaceutical substances. There is a need of commercially production and distribution of effective but inexpensive therapeutic, preventive, and inspective medicines for malaria.

The compound "mefloquine" developed during the Vietnam War is the most commonly used antimalarial drug at present. The newer antimalarial drug is Malarone approved in the US in 2000. This is, however, only the diversion of a known substance to the therapeutic medicine for malaria. The latest study has proposed an inexpensive synthetic compound OZ227 having the similar action mechanism to that of a known natural therapeutic agent `artemisinin` (see Non-Patent Reference 1).

The conventional anti-Plasmodium falciparum drugs, however, generally have severe side effects including headache and nausea. Administration of such medicines for the preventive purpose is thus not generally recommendable. Some of the conventional antimalarial substances, for example, quinine and chloroquine, are deleterious. Peptide vaccines mainly composed of amino acids, on the other hand, exert only desired preventive immunological effects, while being less poisonous and deleterious than the conventional antimalarial substances.

The disadvantage of the peptide vaccine is the potentially different preventive immunological effects among different individuals. The prevalence rate of malaria is high in the epidemic regions. The simple reduction in the risk of malaria development is thus expected to have sufficient contribution to the decrease in the number of sufferers and the number of deaths.

Over many years, the inventors of the present invention have been occupied in development of peptide vaccines, based on the epidemiologic study in the epidemic regions in combination with the molecular analyses in the laboratory scale. As the result of the extensive studies, the inventors have found that enolase, which is a glycolytic enzyme produced from the Plasmodium falciparum-infected human, functions as a protective immune molecule against malaria and have developed peptide vaccines by taking advantage of such finding.

For example, the inventors noted and examined a partial amino acid sequence of enolase from Plasmodium falciparum shown in SEQ ID NO: 12.

A small amount (several milligram level) of a peptide including this partial amino acid sequence (SEQ ID NO: 12) was synthesized and was used as an artificial antigen. The use of this artificial antigen induced an immunological response to Plasmodium falciparum, enabled diagnosis of the immunological state of malaria infection (immunological inspection), and derivatively produced an immunological antigen for inhibiting the proliferation of Plasmodium falciparum. These research results are reported in Patent Reference 1, together with the research results on synthetic peptides comprising other partial amino acid sequences of enolase protein from Plasmodium falciparum.

The peptide vaccine is conventionally produced by the solid-phase synthesis or by the genetic recombination. Neither the solid-phase synthesis technique nor the genetic recombination technique requires the specific skill or experience for producing any sequences. The solid-phase synthesis technique, however, generally has a lower synthesis yield for a longer chain substance and gives a relatively poor yield of a final product after purification. The genetic recombination technique also generally gives a poor yield of a final product.

The laboratory scale synthesis of the peptide including the partial amino acid sequence of enolase from Plasmodium falciparum (SEQ ID NO: 12) by the solid-phase synthesis technique gives a yield of only several hundred micrograms to several milligrams. The synthesis of the peptide by the genetic recombination technique with culture of Eschelichia coli in a relatively large (laboratory scale) 2-liter vessel gives a yield of only 1 to 2 milligrams (as the vaccine for only one person). Even the genetic recombination with an industrially largest-level 1000-liter vessel gives a yield of only 500 to 1000 milligrams (as the vaccine for about 500 people).

Another conventionally known technique potentially applicable for synthesis of the partial peptide (SEQ ID NO: 12) is fragment condensation. In this case, synthesis of a fragment having a glutamic acid residue at the terminal is the most important issue to be solved. Introduction of trichloroethyl ester group or another optional ester group into N-.alpha.-protected-L-glutamic-.gamma.-benzyl ester is essential for obtaining a synthesis intermediate N-.alpha.-t-butoxycarbonyl-L-glutamic-.gamma.-benzyl ester-.alpha.-trichloroethyl ester described later in Examples and other optional synthesis intermediates N-.alpha.-protected-L-glutamic-.gamma.-benzyl ester-.alpha.-protected esters. According to Non-Patent Reference 2, esterification of N-.alpha.-protected-L-glutamic-.gamma.-benzyl ester for the purpose of protecting the .alpha.-site carboxylic acid group has a high potential for racemization. It is thus believed in the art that N-.alpha.-protected-L-glutamic-.gamma.-benzyl ester is not suitable for the synthesis of a peptide. There has been no synthesis tried after the report of the Non-Patent Reference 2.

The synthesis of the partial peptide (SEQ ID NO: 12) by the conventional fragment condensation technique in consideration of the potential racemization requires large fragments having at least 14 residues. The desired size of each fragment is generally 5 to 7 residues at the maximum for the good yields of synthesis and purification. Namely the conventional fragment condensation technique is not adequate for the efficient large-scale synthesis of the peptide.

As used herein: Patent Reference 1: Japanese Patent Laid-Open Gazette No. 2002-371098; Non-Patent Reference 1: pages 900-903, vol. 430, 2004, Nature; and Non-Patent Reference 2: pages 1962-1965, vol. 47, 1982, Journal of Organic Chemistry.

DISCLOSURE OF THE INVENTION

The present invention provides a method that is suitable for large-scale synthesis of a peptide required for inducing an immunological response to Plasmodium falciparum by taking advantage of an immunological reaction in human or another animal. More specifically, the present invention is directed to a method that is suitable for large-scale syntheses of an immunological antigen sequence that is used for inhibiting the proliferation of Plasmodium falciparum and for diagnosing the immunological state of malaria infection.

The inventors of the present invention noted and intensively studied the chemical syntheses in the homogeneous reaction system known as the fragment condensation and planned divisional syntheses of five segments. The examination target was especially placed on the syntheses of two fragments having glutamic acid residues at their terminals, that is, the peptides (II) Phe-Tyr(R.sub.3)-Asn(R.sub.4)-Ser(R.sub.5)-Glu(R.sub.6) (SEQ ID NO: 10) and (I) Xaa-Ala-Ser(R.sub.1) -Glu(R.sub.2) (SEQ ID NO: 11). Based on such examinations, the inventors eventually succeeded in divisional syntheses of the five segments.

Among the fragments, the conventionally unavailable synthesis intermediate N-.alpha.-protected-L-glutamic-.gamma.-benzyl ester-.alpha.-protected ester was used as the starting material for the peptides (I) and (II). We thoroughly examined the synthesis conditions and surprisingly managed to obtain the non-racemized L-form product of the high purity with high efficiency.

Then, we tried to condense the five fragments sequentially or to condense after linking to some larger partial peptides. The inventors examined the optimum condensing agent and eventually succeeded in condensing the five fragments into one protected peptide chain.

The inventors have developed a method that enables a large-scale synthesis of a peptide having a partial sequence of enolase from Plasmodium or its analog by synthesizing an N-x-protected-L-glutamic-.gamma.-benzyl ester-.alpha.-protected ester, synthesizing five segments and condensing the five segments into one protected peptide chain, and thus completed the present invention. Here the terminology `large-scale` implies production of 100-fold or greater scale than the conventional genetic recombination. Even one cycle of laboratory scale synthesis by the method of the present invention yields approximately 100 to 500 milligrams of the peptide (as the vaccine for about 50 to 250 people). Only two to four cycles of the laboratory scale synthesis has the peptide production capacity equivalent to that of industrial genetic recombination equipment of the world-largest scale. Industrial scale synthesis by the method of the present invention is expected to yield the peptide vaccine for several million people, which satisfies the annual global demand.

The present invention includes:

A method for producing a peptide having an amino acid sequence of Xaa Ala Ser Glu Phe Tyr Asn Ser Glu Asn Lys Thr Tyr Asp Leu Asp Phe Lys Thr Pro Asn Asn Asp Xaa (SEQ ID NO: 1), including linking the following fragments (i) through (v) to produce said peptide: (v) Asn-Asn-Asp-Xaa (SEQ ID NO: 2); (iv) Asp-Phe-Lys-Thr-Pro (SEQ ID NO: 3); (iii) Asn-Lys-Thr-Tyr-Asp-Leu (SEQ ID NO: 4); (ii) Phe-Tyr-Asn-Ser-Glu (SEQ ID NO: 5); and (i) Xaa-Ala-Ser-Glu (SEQ ID NO: 6), where `Xaa` in (i) and (v) represents zero or any arbitrary number of amino acid residues.

(2) The production method according to (1), wherein the peptide of SEQ ID NO: 1 is produced by linking the modified peptides (I) through (V) shown below and performing subsequent deprotection: (V)Asn(R.sub.15)Asn(R.sub.16)-Asp(R.sub.17)-Xaa(SEQ ID NO: 7) where R.sub.15 and R.sub.16 represent (C.sub.6H.sub.5).sub.3C-- or no protecting group, and R.sub.17 represents C.sub.6H.sub.5CH.sub.2--O-- or or (CH.sub.3).sub.3C--O--; (IV) Asp(R.sub.12)-Phe-Lys(R.sub.13)-Thr(R.sub.14)-Pro (SEQ ID NO: 8) where R.sub.12 represents C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--, R.sub.13 represents (CH.sub.3).sub.3C--O--CO--, C.sub.6H.sub.5CH.sub.2--O--CO--, 2-chlorobenzyloxycarbonyl- or 9-fluorenylmethoxycarbonyl-, and R.sub.14 represents C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--; (III) Asn(R.sub.7)-Lys(R.sub.8)-Thr(R.sub.9)-Tyr(R.sub.10)-Asp(R.sub.11)-Leu (SEQ ID NO: 9) where R.sub.7 represents (C.sub.6H.sub.5).sub.3C-- or no protecting group, R.sub.8 represents (CH.sub.3).sub.3C--O--CO--, C.sub.6H.sub.5CH.sub.2--O--CO--, 2-chlorobenzyloxycarbonyl- or 9-fluorenylmethoxycarbonyl-, R.sub.9 represents C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--, R.sub.10 represents C.sub.6H.sub.5--CH.sub.2--, Cl.sub.2--C.sub.6H.sub.3--CH.sub.2--, or (CH.sub.3).sub.3C--, and R.sub.11 represents C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--; (II) Phe-Tyr(R.sub.3)-Asn(R.sub.4)-Ser(R.sub.5)-Glu(R.sub.6) (SEQ ID NO: 10) where R.sub.3 represents C.sub.6H.sub.5--CH.sub.2--, Cl.sub.2--C.sub.6H.sub.3--CH.sub.2--, or (CH.sub.3).sub.3C--, R.sub.4 represents (C.sub.6H.sub.5).sub.3C-- or no protecting group, R.sub.5 represents C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--, and R.sub.6 represents C.sub.6H.sub.5CH.sub.2--O-- or (CH.sub.3).sub.3C--O--; and (I) Xaa-Ala-Ser(R.sub.1)-Glu(R.sub.2) (SEQ ID NO: 11) where R.sub.1 represents C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--, and R.sub.2 represents C.sub.6H.sub.5CH.sub.2--O-- or (CH.sub.3).sub.3C--O--.

(3) The production method according to items 1 or 2, wherein said peptides are condensed by using a combination of 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1 -hydroxybenzotriazole, a combination of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazol, or O-(7-azabenzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate.

(4) A method for producing a peptide of SEQ ID NO: 1 having a modification in its terminus, including: producing the peptide of SEQ ID NO: 1 by the method according to any one of (1) to (3), and adding a sugar chain sequence, a peptide, a protein, a polysaccharide, a metal complex, a polymer carrier, a gel, a film, latex particles, metal fine particles, or a plastic plate to an N terminus and/or a C terminus of the peptide of SEQ ID NO: 1.

(5) A method for manufacturing a preventive or therapeutic medicine for Plasmodium falciparum infection, or a diagnostic agent for Plasmodium falciparum infection, including: producing the peptide of SEQ ID NO: 1 by the method according to one of (1) to (3); and formulating the produced peptide of SEQ ID NO: 1 with a pharmaceutically acceptable carrier.

(6) A method of manufacturing a preventive or therapeutic medicine for Plasmodium falciparum infection, or a diagnostic agent for Plasmodium falciparum infection, including: producing the terminal-modified peptide of SEQ ID NO: 1 by the method of (4); and formulating the terminal-modified peptide of SEQ ID NO: 1 with a pharmaceutically acceptable carrier.

(7) N-.alpha.-t-butoxycarbonyl-L-glutamic-.gamma.-benzyl-.alpha.-trichloro- ethyl ester that essentially consists of an L-form.

(8) A method for producing a peptide including using the N-.alpha.-t-butoxycarbonyl-L-glutamic-.gamma.-benzyl-.alpha.-trichloroeth- yl ester according to item (7).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the present invention, the fragments (i) through (v) shown below are linked to produce a peptide having an amino acid sequence of Xaa Ala Ser Glu Phe Tyr Asn Ser Glu Asn Lys Thr Tyr Asp Leu Asp Phe Lys Thr Pro Asn Asn Asp Xaa (SEQ ID NO: 1): (v) Asn-Asn-Asp-Xaa (SEQ ID NO: 2); (iv) Asp-Phe-Lys-Thr-Pro (SEQ ID NO: 3); (iii) Asn-Lys-Thr-Tyr-Asp-Leu (SEQ ID NO: 4); (ii) Phe-Tyr-Asn-Ser-Glu (SEQ ID NO: 5); and (i) Xaa-Ala-Ser-Glu (SEQ ID NO: 6), where `Xaa` in the (i) and (v) represents zero, one, or plural, that is, any arbitrary number of, amino acid residues. The number of the amino acid residues included in `Xaa` is not specifically restricted, as long as the resulting peptide of SEQ ID NO: 1 has the ability of inducing an immunological response to Plasmodium falciparum. The preferable number of the amino acid residues included in `Xaa` is in the range of 0 to 20. In the (i) and (v), `Xaa` may further include a carrier, in addition to the amino acid residues.

The peptides (i) through (v) may be produced by any conventional peptide synthesis technique but are preferably obtained by the solution phase reaction.

The peptide (ii) and the peptide (i) with Xaa=zero amino acid residue have L-glutamic acid at their C terminals. The conventionally unavailable N-.alpha.-protected-L-glutamic-.gamma.-benzyl ester-.alpha.-protected ester or more specifically N-.alpha.-t-butoxycarbonyl-L-glutamic-.gamma.-benzyl-.alpha.-trichloroeth- yl ester produced by the inventors of the present invention is preferably used as the reaction starting material for syntheses.

For production of the peptide of SEQ ID NO: 1, the peptides (i) through (v) may be linked sequentially one by one from one end peptide (i) or from the other end peptide (v), that is, from the fragment at the N-terminal or from the fragment at the C-terminal of the peptide of SEQ ID NO: 1. Another available procedure may first link some of the peptides, for example, (i) and (ii), to one larger fragment and the others of the peptides, for example, (iii), (iv), and (v), to another larger fragment and link the two larger fragments.

Prior to the linkage of peptide fragments, it is preferable to protect the terminals of the fragments, which are not involved in the linkage reaction, as well as the reactive side chains of these fragments. For example, in the case of linkage of (ii) Asp-Phe-Lys-Thr-Pro (SEQ ID NO: 3) with (iii) Asn-Lys-Thr-Tyr-Asp-Leu (SEQ ID NO: 4), the synthesis procedure preferably protects Asp at the amino terminal of (ii), Leu at the carboxyl terminal of (iii), and all the reactive side chains of these peptides.

Preferable examples of the protecting group at the amino terminal include (CH.sub.3).sub.3C--O--CO--, C.sub.6H.sub.5CH.sub.2--O--CO--, and 9-fluorenylmethoxycarbonyl-. Preferable examples of the protecting group at the carboxyl terminal include --O--CH.sub.2--CCl.sub.3, --O--CH.sub.2--CO--C.sub.6H.sub.5, and --O--CH.sub.2--C.sub.6H.sub.5.

The protecting groups for protection of the reactive side chains are appropriately selected depending on the amino acids. The following shows examples of side chain-protected fragments (i) through (v): (V) Asn(R.sub.15)-Asn(R.sub.16)-Asp(R.sub.17)-Xaa(SEQ ID NO: 7) where R.sub.15 and R.sub.16 represent side chain protecting groups of asparagine residue (for example, (C.sub.6H.sub.5).sub.3C--) or no protecting group, and R.sub.17 represents a side chain protecting group of aspartic acid group (for example, C.sub.6H.sub.5CH.sub.2--O-- or (CH.sub.3).sub.3C--O--); (IV) Asp(R.sub.12)-Phe-Lys(R.sub.13)-Thr(R.sub.14)-Pro(SEQ ID NO: 8) where R.sub.12 represents a side chain protecting group of aspartic acid group (for example, C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--), R).sub.3 represents a side chain protecting group of lysine residue (for example, (CH.sub.3).sub.3C--O--CO--, C.sub.6H.sub.5CH.sub.2--O--CO--, 2-chlorobenzyloxycarbonyl- or 9-fluorenylmethoxycarbonyl-), and R.sub.14 represents a side chain protecting group of threonine residue (for example, C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--); (III) Asn(R.sub.7)-Lys(R.sub.8)-Thr(R.sub.9)-Tyr(R.sub.10)-Asp(R.sub.11)-Leu (SEQ ID NO: 9) where R.sub.7 represents a side chain protecting group of asparagine residue (for example, (C.sub.6H.sub.5).sub.3C--) or no protecting group, R.sub.8 represents a side chain protecting group of lysine residue (for example, (CH.sub.3).sub.3C--O--CO--, C.sub.6H.sub.5CH.sub.2--O--CO--, 2-chlorobenzyloxycarbonyl- or 9-fluorenylmethoxycarbonyl-), R.sub.9 represents a side chain protecting group of threonine residue (for example, C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--), R.sub.10 represents a side chain protecting group of tyrosine residue (for example, C.sub.6H.sub.5--CH.sub.2--, Cl.sub.2--C.sub.6H.sub.3--CH.sub.2--, or (CH.sub.3).sub.3C--), and R.sub.11 represents a side chain protecting group of aspartic acid group (for example, C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--); (II) Phe-Tyr(R.sub.3)-Asn(R.sub.4)-Ser(R.sub.5)-Glu(R.sub.6) (SEQ ID NO: 10) where R.sub.3 represents a side chain protecting group of tyrosine residue (for example, C.sub.6H.sub.5--CH.sub.2--, Cl.sub.2-C.sub.6H.sub.3--CH.sub.2--, or (CH.sub.3).sub.3C--), R.sub.4 represents a side chain protecting group of asparagine residue (for example, (C.sub.6H.sub.5).sub.3C-- or no protecting group), R.sub.5 represents a side chain protecting group of serine residue (for example, C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--), and R.sub.6 represents a side chain protecting group of aspartic acid group (for example, C.sub.6H.sub.5CH.sub.2--O-- or (CH.sub.3).sub.3C--O--) (I) Xaa-Ala-Ser(R.sub.1)-Glu(R.sub.2) (SEQ ID NO: 11) where R.sub.1 represents a side chain protecting group of serine residue (for example, C.sub.6H.sub.5CH.sub.2-- or (CH.sub.3).sub.3C--), and R.sub.2 represents a side chain protecting group of glutamic acid residue (for example, C.sub.6H.sub.5CH.sub.2--O-- or (CH.sub.3).sub.3C--O--).

The linkage of the respective fragments follows a conventional peptide condensation reaction, preferably using a condensing agent. Desirable examples of the condensing agent include a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole, a combination of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazol, or O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate alone. The condensation reaction is preferably performed in the solution phase for the enhanced yield.

Deprotection of the linked peptides yields the peptide of SEQ ID NO: 1. The deprotection follows a conventionally known method.

For the easy induction of an immunological reaction, the peptide of the present invention is desirably designed and produced to stabilize the high-order structure of enolase from Plasmodium falciparum. The terminology "immunological response" in the specification hereof is the concept including both cellular immunological responses and humoral immunological responses. The "cellular immunological responses" means immunological responses caused by, for example, macrophages, natural killer cells (NK cells), eosinophils, and T cells. The "known cellular immunological response" to Plasmodium falciparum is an immunological response caused by killer T cells. The "known humoral immunological responses" means immunological responses caused by host-derived antibodies that specifically binds to proteins and sugar chains from Plasmodium falciparum. The antigen peptide produced by the present invention desirably has the ability of inducing an antibody as the humoral immunological response.

More specifically, a terminal-modified peptide is preferably produced by introduction of a compound for inducing a high-order structure into at least either of the amino terminal and the carboxyl terminal in the peptide of SEQ ID NO: 1. The high-order structure is easily recognizable by macrophages, NK cells, T cells, and other immunological cells, as well as by antibodies.

Typical examples of the compound for inducing the high-order structure include sugar chain sequences, peptide sequences, proteins, polysaccharides, metal complexes, polymer carriers, gels, films, latex particles, metal fine particles, and plastic plates.

Any of these modifier compounds may be introduced to either or both of the amino terminal and the carboxyl terminal in the peptide of SEQ ID NO: 1 by a bond corresponding to the type of the modifier compound, for example, by covalent bond, ionic bond, or coordination bond.

A peptide-bound film is prepared, for example, by the spin cast method. The presence of an antibody included in a test sample is detectable by placing dots of the test sample on the prepared peptide-bound film. Peptide-bound latex particles are prepared, for example, by emulsion polymerization or by suspension polymerization. The prepared peptide-bound latex particles are usable for aggregation reactions. A peptide-bound plastic plate is prepared, for example, by placing an adequate number of drops of the peptide in wells of the plastic plate. Peptide-bound microbeads are prepared, for example, by soaking microbeads in a peptide solution.

The peptide production method of the present invention is also applicable to prepare a substance including a plurality of the peptide sequence in its molecular structure. A linker may be used to link the plurality of the peptide to produce the substance including the plurality of the peptide. The linker works to join the plurality of the peptide sequence in a linear arrangement (see FIG. 6 (see Original Patent)) or in a branched arrangement to form a high molecule. The number of the peptide sequence to be linked is not restricted but is preferably in a range of 4 to 8.

In FIG. 8 (see Original Patent), AD22 represents the peptide of SEQ ID NO: 1 or the terminal-modified peptide that is introduced with the compound for inducing the high-order structure at least either of the amino terminal and the carboxyl terminal in the peptide of SEQ ID NO: 1. The dotted line represents the repeated linear linkage of the peptides.

The linker may be one or a combination selected among amino acid sequences, sugar chain sequences, dicarboxlates, diamines, and metal complexes, that have covalent bond, ionic bond, and coordination bond, although these examples are not restrictive. The linker may be a peptide corresponding to Xaa in the peptide sequence of SEQ ID NO: 1 or the compound to be linked to Xaa for inducing the high-order structure.

Typical examples of the carrier molecule or polymer carrier are various natural proteins including tetanus toxoid, ovalbumin, serum albumin, and hemocyanin.

The peptide or the terminal-modified peptide produced by the method of the present invention or the compound including the plurality of the peptides linked to each other is formulated with a pharmaceutically acceptable carrier to produce a preventive medicine or a therapeutic medicine for Plasmodium falciparum infection or a diagnostic agent for Plasmodium falciparum infection. The terminology "pharmaceutically acceptable carrier" in the specification hereof includes, for example, immunostimulators, diluents, stabilizers, preservatives, and buffers.

The preventive medicine or the therapeutic medicine for Plasmodium falciparum infection may be, for example, a vaccine for preventing malaria infection or a treatment vaccine for stimulating the immune system of the malaria-infected patient against the antigen from Plasmodium falciparum. The diagnostic agent for Plasmodium falciparum infection may be a diagnostic agent for the presence of an antibody against the antigen from Plasmodium falciparum.

The method of the present invention may be adopted to produce an analog of the peptide of SEQ ID NO: 1. The terminology "analog of the peptide" means a peptide that is obtained by introducing substitution, deletion, or insertion of one or multiple amino acids in the peptide of SEQ ID NO: 1 and has a similar immunological response to that of the peptide of the present invention. The number of amino acids substituted, deleted, or inserted is not specifically restricted but is preferably in a range of 1 to 5 and more specifically in a range of 1 to 2.

The analog sequence may be used to enhance the solubility and the crystalline property in chemical syntheses or to enhance the solubility and the immunological response in immunological reactions, although these applications are not restrictive. The analog sequence may also be used for production of preventive, therapeutic, and diagnostic medicines.

The present invention is also directed to N-.alpha.-t-butoxycarbonyl-L-glutamic-.gamma.-benzyl-.alpha.-trichloroeth- yl ester (Boc-Glu(OBzl)-OTce) that essentially consists of the L-form and is used for synthesis of the peptide of SEQ ID NO: 1. It is conventionally believed in the art that synthesis of the pure L-form Boc-Glu(OBzl)-OTce is practically impossible because of the potential racemization (Non-Patent Reference 2: pages 1962-1965, vol. 47, 1982, Journal of Organic Chemistry). The inventors of the present invention have managed to yield substantially pure L-form Boc-Glu(OBzl)-OTce by the DCC condensation reaction of N-.alpha.-t-butoxycarbonyl-L-glutamic-.gamma.-benzyl ester and trichloroethyl alcohol in the presence of a catalyst DMAP as a reaction accelerator at a molar ratio of only 0.1 equivalent, which is significantly less than the conventional molar ratio of 0.5 equivalent. The L-form Boc-Glu(OBzl)-OTce is usable for syntheses of various glutamic acid-containing peptides, as well as the peptide of SEQ ID NO: 1. The terminology "essentially consisting of the L-form" means the L-form of not less than 95%, preferably not less than 98%, more preferably not less than 99%, or most preferably equal to 100%.
 

Claim 1 of 18 Claims

1. A method for producing a peptide having an amino acid sequence of Xaa Ala Ser Glu Phe Tyr Asn Ser Glu Asn Lys Thr Tyr Asp Leu Asp Phe Lys Thr Pro Asn Asn Asp Xaa (SEQ ID NO: 1), comprising linking the following fragments (i) through (v) to produce said peptide: (v) Asn-Asn-Asp-Xaa (SEQ ID NO: 2); (iv) Asp-Phe-Lys-Thr-Pro (SEQ ID NO: 3); (iii) Asn-Lys-Thr-Tyr-Asp-Leu (SEQ ID NO: 4); (ii) Phe-Tyr-Asn-Ser-Glu (SEQ ID NO: 5); and (i) Xaa-Ala-Ser-Glu (SEQ ID NO: 6), where `Xaa` in (i) and (v) represents zero or any arbitrary number of amino acid residues.

 

 

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