JP2019090030A - Isoprene oligomer, polyisoprene, and method for producing the same, rubber composition, and pneumatic tire - Google Patents

Abstract

To provide an isoprene oligomer and a polyisoprene with a modified main chain of a molecule, and a rubber composition containing the isoprene oligomer and/or the polyisoprene, and a pneumatic tire with the rubber composition used for each member of a tire.SOLUTION: The present invention provides an isoprene oligomer synthesized from an allyl diphosphoric acid represented by a formula (X) and a compound represented by a formula (Y) (n is an integer of 1-10, R is a group other than a methyl group).SELECTED DRAWING: None

Description

The present invention relates to an isoprene oligomer, a polyisoprene, and a method for producing them, a rubber composition containing isoprene oligomer and / or polyisoprene, and a pneumatic tire using the rubber composition.

Heretofore, in rubber products, fillers of various materials and shapes are introduced into rubber compositions according to their applications, for the purpose of imparting new properties to the properties inherently possessed by rubber. By doing this, it is practiced to express desired characteristics. For example, in automobile tires, fillers such as silica and carbon black are introduced into the rubber phase which is an organic substance to improve the properties such as wear resistance, low heat buildup and wet grip performance. .

In mixing such a filler with a rubber phase in such a rubber composition, the rubber contained in the rubber phase for the purpose of enhancing the affinity between the two and further improving the low heat buildup and wet grip performance etc. The molecule is treated, for example, by reacting a compound having a nitrogen atom-containing group and a chlorosulphenyl group to introduce a functional group exhibiting affinity to the filler into the rubber molecule. It has been practiced to use a modified rubber (modified diene polymer) (for example, Patent Documents 1 and 2).

Also, it is known that highly reactive diphosphate groups etc. are present at the terminal part of isoprene oligomer or polyisoprene. Therefore, when modifying an isoprene oligomer or polyisoprene, a compound having a functional group is reacted with these groups by using a highly reactive diphosphate group or the like present at the terminal portion. Denaturation has been performed. Thus, conventionally, modifications to isoprene oligomers and polyisoprene have mainly been performed on the terminal portion of the molecule. On the other hand, in the case of modifying the main chain portion of isoprene oligomer or polyisoprene, the main chain portion could not be sufficiently modified due to the influence of the highly reactive diphosphate group present at the terminal portion. Also, in the case of modifying natural rubber up to now, there is only a method of modifying the latex collected from Hevea brasiliensis by chemical treatment, and what functional group conversion is actually whether the molecular chain has been modified. It was analytically impossible to confirm what happened.

JP 2000-001573 A JP 2000-001575 A

An object of the present invention is to solve the above-mentioned problems, and to provide an isoprene oligomer, polyisoprene, in which a main chain portion of a molecule is modified. Another object of the present invention is to provide a rubber composition containing the isoprene oligomer and / or the polyisoprene, and a pneumatic tire using the rubber composition for each component (for example, tread, sidewall) of a tire. .

（式（Ｘ）中、ｎは１〜１０の整数を表す。）

（式（Ｙ）中、Ｒはメチル基以外の基を表す。） The present invention relates to an isoprene oligomer obtained by synthesizing from allylic diphosphate represented by the following formula (X) and a compound represented by the following formula (Y).

(In the formula (X), n represents an integer of 1 to 10)

(In formula (Y), R represents a group other than a methyl group.)

The isoprene oligomer is preferably obtained by being synthesized from allylic diphosphate represented by the formula (X), a compound represented by the formula (Y), and isopentenyl diphosphate.

（式（Ｘ−１）中、ｎは１〜１０の整数を表す。） An allylic diphosphate represented by the above formula (X) is an allylic diphosphate represented by the following formula (X-1), and an atom contained in a portion II in the following formula (X-1) Or at least one of the atomic groups is substituted by another atom or atomic group, and preferably the atom or atomic group contained in the portion III in the following formula (X-1) is not substituted by another atom or atomic group .

(In the formula (X-1), n represents an integer of 1 to 10)

It is preferable to carry out the above synthesis using an enzyme having prenyltransferase activity.

（式（Ｚ−１）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。）

（式（Ｚ−２）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。） The present invention is also an isoprene oligomer represented by the following formula (Z-1) or the following formula (Z-2), wherein v moiety in the following formula (Z-1) or the following formula (Z-2) It relates to an isoprene oligomer in which at least one of the contained atoms or atomic groups is substituted by another atom or atomic group.

(In the formula (Z-1), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

(In the formula (Z-2), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxy carboxy group, an alkoxy carbonyl group or an OPP group Represents

It is preferable that at least one of the atom or atomic group contained in the iv part in the said Formula (Z-1) or said Formula (Z-2) is substituted by the other atom or atomic group.

（式（Ｘ）中、ｎは１〜１０の整数を表す。）

（式（Ｙ）中、Ｒはメチル基以外の基を表す。） The present invention also relates to a method for producing an isoprene oligomer synthesized from allylic diphosphate represented by the following formula (X) and a compound represented by the following formula (Y).

(In the formula (X), n represents an integer of 1 to 10)

(In formula (Y), R represents a group other than a methyl group.)

The method for producing the isoprene oligomer is preferably synthesized from allylic diphosphate represented by the formula (X), a compound represented by the formula (Y), and isopentenyl diphosphate.

（式（Ｘ−１）中、ｎは１〜１０の整数を表す。） An allylic diphosphate represented by the above formula (X) is an allylic diphosphate represented by the following formula (X-1), and an atom contained in a portion II in the following formula (X-1) Or at least one of the atomic groups is substituted by another atom or atomic group, and preferably the atom or atomic group contained in the portion III in the following formula (X-1) is not substituted by another atom or atomic group .

(In the formula (X-1), n represents an integer of 1 to 10)

It is preferable to carry out the above synthesis using an enzyme having prenyltransferase activity.

（式（Ｘ）中、ｎは１〜１０の整数を表す。）

（式（Ｙ）中、Ｒはメチル基以外の基を表す。） The present invention also relates to a polyisoprene obtained by synthesizing from allylic diphosphate represented by the following formula (X) and a compound represented by the following formula (Y).

(In the formula (X), n represents an integer of 1 to 10)

(In formula (Y), R represents a group other than a methyl group.)

The compound is preferably obtained by synthesis from allylic diphosphate represented by the above formula (X), a compound represented by the above formula (Y), and isopentenyl diphosphate.

（式（Ｘ−１）中、ｎは１〜１０の整数を表す。） An allylic diphosphate represented by the above formula (X) is an allylic diphosphate represented by the following formula (X-1), and an atom contained in a portion II in the following formula (X-1) Or at least one of the atomic groups is substituted by another atom or atomic group, and preferably the atom or atomic group contained in the portion III in the following formula (X-1) is not substituted by another atom or atomic group .

(In the formula (X-1), n represents an integer of 1 to 10)

The present invention also relates to a polyisoprene obtained by synthesizing the above isoprene oligomer and the compound represented by the above formula (Y) and / or isopentenyl diphosphate.

（式（ＺＺ−１）中、ｎは１〜１０の整数を表す。ｑは３０〜４００００の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。）

（式（ＺＺ−２）中、ｎは１〜１０の整数を表す。ｑは３０〜４００００の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。） The present invention is also a polyisoprene represented by the following formula (ZZ-1) or the following formula (ZZ-2), wherein v moiety in the following formula (ZZ-1) or the following formula (ZZ-2) It relates to polyisoprene in which at least one of the contained atoms or atomic groups is substituted by another atom or atomic group.

(In the formula (ZZ-1), n represents an integer of 1 to 10. q represents an integer of 30 to 40. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

(In the formula (ZZ-2), n represents an integer of 1 to 10. q represents an integer of 30 to 40. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

It is preferable that at least one atom or atomic group contained in the iv moiety in the above formula (ZZ-1) or the above formula (ZZ-2) is substituted by another atom or atomic group.

The present invention also relates to a method for producing polyisoprene synthesized from the above isoprene oligomer, and the compound represented by the above formula (Y) and / or isopentenyl diphosphate.

The present invention also relates to a rubber composition comprising the above isoprene oligomer and / or the above polyisoprene.

The present invention also relates to a pneumatic tire produced using the above rubber composition.

The isoprene oligomer of the present invention is an isoprene oligomer obtained by being synthesized from allylic diphosphate represented by the above formula (X) and a compound represented by the above formula (Y), or the above formula (Z-1) Or an isoprene oligomer represented by the above formula (Z-2), wherein at least one atom or atomic group contained in the v moiety in the above formula (Z-1) or the above formula (Z-2) is It is an isoprene oligomer substituted by another atom or group. In addition, the polyisoprene of the present invention is a polyisoprene obtained by synthesis from allylic diphosphate represented by the above formula (X) and a compound represented by the above formula (Y), the above isoprene oligomer, Polyisoprene obtained by being synthesized from the compound represented by the above formula (Y) and / or isopentenyl diphosphate, or polyisoprene represented by the above formula (ZZ-1) or the above formula (ZZ-2) A polyisoprene in which at least one of the atoms or atomic groups contained in the v moiety in the above formula (ZZ-1) or the above formula (ZZ-2) is substituted by another atom or atomic group . Therefore, the isoprene oligomer of the present invention and the polyisoprene of the present invention are modified in the main chain portion of the molecule (rubber molecule), and are excellent in the affinity to the filler such as silica. Therefore, by blending the isoprene oligomer of the present invention and / or the polyisoprene of the present invention into a rubber composition, a rubber composition in which rubber molecules and a filler are combined at a high level can be obtained. It is possible to provide a rubber composition excellent in abrasion resistance. In addition, by using the rubber composition for each component (for example, a tread, a sidewall) of a tire, for example, a pneumatic tire excellent in low heat buildup and abrasion resistance can be provided.
Further, in the method for producing isoprene oligomer of the present invention and the method for producing polyisoprene of the present invention, the main chain portion is sufficiently modified without being affected by the highly reactive diphosphate group present at the terminal portion. Isoprene oligomers and polyisoprene can be produced.
Further, in the present invention, since the isoprene oligomer obtained by using the compound represented by the above-mentioned formula (Y) to which a functional group or the like with a known structure is added is polyisoprene, the isoprene oligomer and the polyisoprene have a function The group (modified group) can be clearly understood.

In the step of artificially synthesizing (biosynthesizing) a rubber molecule (polyisoprene), an enzyme such as prenyltransferase acts on a mixture of an initiating substrate such as farnesyl diphosphate (FPP) and a monomer such as isopentenyl diphosphate. By doing this, an isoprene oligomer in which about 1 to 30 isoprene units are addition-polymerized with respect to the starting substrate is formed. Thereafter, by mixing a latex component containing an enzyme which addition-polymerizes isopentenyl diphosphate with the isoprene oligomer, a polyisoprene in which a large number of isopentenyl diphosphates are linked to the oligomer is formed. It has been known.

Thus, addition polymerization by a natural enzyme is indispensable in each process of sequentially bonding a monomer to a starting substrate to make a rubber molecule.

For this reason, as a starting substrate or a monomer for synthesizing (biosynthesizing) a rubber molecule (polyisoprene), it is necessary to use one which catalyzes a reaction of the enzyme to be used, and as a result, it is possible to use rubber molecules (polyisoprene). The structures of starting substrates and monomers used as raw materials were limited. In the case of monomers, in particular, due to the restriction caused by the addition polymerization of prenyltransferase and isopentenyl diphosphate, it was limited to naturally occurring isopentenyl diphosphate.

As a result, even in a rubber molecule (polyisoprene) artificially synthesized (biosynthesized), the degree of freedom in its structure is limited, and it is difficult to design free molecules for adding functionality not found in natural rubber. The

Therefore, for example, when it is desired to obtain a rubber molecule (polyisoprene) into which a functional group or the like has been introduced, as described above, a functional highly functional diphosphate group or the like present at the terminal portion is used to obtain a functional Modification has been carried out by reacting compounds having groups with these groups. In particular, in the case of modifying natural rubber, there is only a method of modifying the latex collected from Hevea brasiliensis by chemical treatment, and what functional group conversion has taken place, indeed, the molecular chain has been modified. It was analytically impossible to confirm the

On the other hand, according to the present invention, an isoprene oligomer having functionality added to the main chain by using isopentenyl diphosphate in which a part of the structure is modified as a monomer for producing an isoprene oligomer or polyisoprene. And polyisoprene was found to be possible. Further, in the present invention, since it is a polyisoprene which is an isoprene oligomer obtained by using isopentenyl diphosphate to which a functional group having a known structure and the like is added, a functional group (modified group) possessed by isoprene oligomer and polyisoprene. Can be clearly understood.

In particular, the present invention relates to the case where the methyl group at position 3 is substituted with a desired group by maintaining the structure of a moiety other than the methyl group at position 3 of the naturally occurring monomer isopentenyl diphosphate. Even by using prenyltransferase, which is a naturally occurring oligomer-forming enzyme, and an enzyme which addition-polymerizes isopentenyl diphosphate, which is a naturally occurring polymer-forming enzyme, isoprene oligomers and polyisoprene can be produced. It is based on having found that. The reason for this is not necessarily clear, but an enzyme that adds and polymerizes prenyltransferase and isopentenyl diphosphate causes adsorption to the structure of the portion other than the methyl group at position 3 of the monomer isopentenyl diphosphate, and the position 3 It is believed that the structure of the methyl group portion of is relatively insensitive.

Based on the findings, it becomes possible to provide an isoprene oligomer or polyisoprene having a main chain having desired properties, and it is possible to provide an isoprene oligomer or polyisoprene having various functions.

That is, in biosynthesis performed in nature, geranyl diphosphate (GPP) is obtained, for example, by sequentially polymerizing isopentenyl diphosphate (IPP) with dimethylallyl diphosphate (DMAPP) as described below. , Farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and geranyl farnesyl diphosphate (GFPP).

On the other hand, in the present invention, instead of isopentenyl diphosphate (IPP), R-IPP in which the methyl group present at the 3-position of IPP is substituted with a desired group (-R) is used as follows: Further, geranyl diphosphate (R-GPP) whose main chain is modified by sequentially polymerizing R-IPP to dimethylallyl diphosphate, farnesyl diphosphate (R-FPP) whose main chain is modified, The main chain is modified to form geranylgeranyl diphosphate (R-GGPP), and the main chain is modified to geranyl farnesyl diphosphate (R-GFPP).

Moreover, by using R-IPP together with IPP, that is, by using R-IPP and IPP in combination, by sequentially polymerizing IPP and R-IPP to dimethylallyl diphosphate as described below Main chain modified geranyl diphosphate (R-GPP) main chain modified farnesyl diphosphate (R-FPP) main chain modified geranyl geranyl diphosphate (R-GGPP) mainly Chain modified geranyl farnesyl diphosphate (R-GFPP) is produced. Thus, isoprene oligomers having desired properties can be prepared by using IPP in combination with R-IPP and adjusting the ratio.

In the present specification, to modify the main chain of a molecule (rubber molecule) means to modify the structure corresponding to IPP present in the main chain of the molecule (rubber molecule) (in particular, the methyl group present at position 3 of IPP) The corresponding moiety) is substituted with the desired functional group, or the structural moiety corresponding to IPP present in the main chain of the molecule (rubber molecule) (especially corresponding to the methyl group present at the 3-position of IPP) Part) is substituted with a different structure (structure other than a methyl group).

（式（Ｘ）中、ｎは１〜１０の整数を表す。）

（式（Ｙ）中、Ｒはメチル基以外の基を表す。） (Isoprene oligomer)
The isoprene oligomer of the present invention is obtained by synthesis (biosynthesis) from allylic diphosphate represented by the following formula (X) and a compound (R-IPP) represented by the following formula (Y) It is an oligomer.

(In the formula (X), n represents an integer of 1 to 10)

(In formula (Y), R represents a group other than a methyl group.)

In the present specification, OPP (OPP group) means a diphosphate group (a group represented by the following formula (A-1)) and has three hydroxyl groups bonded to a phosphorus atom However, in the aqueous solution, some or all of these hydroxyl groups dissociate (for example, it becomes a group represented by the following formula (A-2)). In the present specification, OPP is a concept also including a group in which part or all of such hydroxyl groups are dissociated.

The isoprene oligomer of the present invention has a structure close to that of natural rubber and has high compatibility with rubber molecules. In addition, the isoprene oligomer of the present invention has a strong interaction with a filler such as silica since the main chain portion is modified. Thus, the isoprene oligomer of the present invention is highly compatible with the rubber, and further, has a strong interaction with the filler such as silica. A rubber composition in which a molecule and a filler are combined can be obtained, and for example, the low heat buildup, wet grip performance, abrasion resistance, elongation at break, and breaking strength of the rubber composition can be improved.

In the isoprene oligomer of the present invention, polar groups and the like are present in the main chain portion. Therefore, compared with the case where it has a polar group etc. only at the end part, the dispersibility of the filler such as silica is high, for example, the improvement effect of low heat buildup, wet grip performance, wear resistance, elongation at break, breaking strength Is high.

In addition, the isoprene oligomer of the present invention exhibits excellent antibacterial activity. This is different in structure from the normal isoprene oligomer which exists in nature, and inhibition of enzyme or coenzyme possessed by bacteria, inhibition of nucleic acid synthesis, inhibition of cell membrane synthesis, inhibition of synthesis of cytoplasmic membrane, disruption of cell membrane, destruction of cytoplasmic membrane It is presumed that this is to have an action such as destruction.

（式（Ｘ）中、ｎは１〜１０の整数を表す。） First, allylic diphosphate represented by the following formula (X) will be described.

(In the formula (X), n represents an integer of 1 to 10)

N of Formula (X) represents an integer of 1 to 10 (preferably 1 to 4, more preferably 1 to 3).

Examples of allylic diphosphates represented by the above formula (X) include dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP). And geranyl farnesyl diphosphate (GFPP). Among them, DMAPP, GPP and FPP are preferred because they are substrates for many prenyltransferases.

The allylic diphosphate represented by the above formula (X) has a structure in which a diphosphate group is bonded to an isoprene unit, but the allylic diphosphate represented by the above formula (X) And ally may be an allylic diphosphate derivative in which a part of the isoprene unit is modified (modified).

The present inventors have already filed patent applications for a method for producing isoprene oligomers and polyisoprenes using an allylic diphosphate derivative as a starting substrate (Japanese Patent Application Laid-Open No. 2012-036360). The patent application is briefly described below.

As described above, as a starting substrate or a monomer for synthesizing (biosynthesizing) a rubber molecule (polyisoprene), it is necessary to use one which catalyzes a reaction of the enzyme to be used, and as a result, a rubber molecule (polyisoprene) The structures of starting substrates and monomers used as the raw materials of (a) were limited. Specifically, the starting substrate is limited to naturally occurring dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranyl geranyl diphosphate, etc. by the restriction caused by the enzyme for producing an oligomer. It had been.

On the other hand, as a result of intensive investigations, the present inventors used farnesyl diphosphate or the like having a partially modified structure as a starting substrate for producing isoprene oligomer or polyisoprene. It has been found that it is possible to produce functional isoprene oligomers and polyisoprene.

In particular, even when the desired structure is introduced into the other part by maintaining the structure of part I of the following formula (I) with respect to the naturally occurring starting substrate farnesyl diphosphate etc. It has been found that isoprene oligomers can be produced by using prenyltransferase, which is a naturally occurring oligomer-forming enzyme. The reason for this is not necessarily clear, but it is considered that prenyltransferase causes adsorption to the structure of the I portion of the following formula (I) of the starting substrate and is relatively insensitive to the structures of other portions.

Based on this finding, it becomes possible to provide an isoprene oligomer or polyisoprene having a terminal end having desired properties, and an isoprene oligomer or isoprene oligomer having various functions added without impairing the properties of the isoprene oligomer or polyisoprene itself. It becomes possible to provide polyisoprene.

Therefore, in the present invention, an allylic diphosphate represented by the above-mentioned formula (X) modified as long as the structure maintains I part of the above-mentioned formula (I) is used as a starting substrate, By polymerizing the compound (R-IPP) represented by the above-mentioned formula (Y) to the starting substrate using a prenyltransferase which is not a main chain portion but also a terminal portion (structure derived from allylic diphosphate) Partially, isoprene oligomer which is also modified is obtained.

（式（Ｘ−１）中、ｎは１〜１０の整数を表す。（式（Ｘ−１）中のｎは、式（Ｘ）中のｎと同一である。）） Specifically, the allylic diphosphate represented by the above formula (X) is an allylic diphosphate represented by the following formula (X-1), and II in the following formula (X-1) At least one of the atoms or atomic groups contained in the moiety is substituted by another atom or atomic group, and the atom or atomic group contained in the III moiety in the following formula (X-1) is substituted by another atom or atomic group Preferably not.

(In the formula (X-1), n represents an integer of 1 to 10. (n in the formula (X-1) is the same as n in the formula (X)))

Thereby, in addition to the main chain portion, an isoprene oligomer having a modified end portion is obtained. Since the isoprene oligomer is modified in addition to the main chain portion and also at the end portion, the interaction with the filler such as silica is further improved, and the isoprene oligomer is further incorporated into the rubber composition than in the prior art. A rubber composition in which rubber molecules and a filler are combined in higher dimensions can be obtained, and for example, the low heat buildup, wet grip performance, abrasion resistance, elongation at break, and breaking strength of the rubber composition can be further improved.

In the present specification, to modify the terminal part of the molecule (rubber molecule) means to modify the structure portion derived from the allylic diphosphate represented by the above formula (X) present at the end of the molecule (rubber molecule). A predetermined portion of the structural portion derived from the allylic diphosphate represented by the above-mentioned formula (X) present at the end of the molecule (rubber molecule) that the predetermined portion is substituted by the desired functional group Is replaced by a different structure.

Examples of the atoms or atomic groups (atoms or atomic groups before being substituted) contained in the portion II and the portion III in the above formula (X-1) include, for example, hydrogen atom, methyl group, methylene group, carbon atom, methine And the like.

As said other atom, a nitrogen atom, an oxygen atom, a sulfur atom, a silicon atom, a carbon atom etc. are mentioned, for example. Among them, a nitrogen atom is preferable because it has strong intermolecular force, causes strong interaction with an enzyme or a cell membrane, and also has strong interaction with a filler such as silica.

Examples of the other atomic groups include nitrogen atom-containing groups, oxygen atom-containing groups, sulfur atom-containing groups, silicon atom-containing groups, carbon atom-containing groups and the like, and examples thereof include an acetoxy group and an alkoxy group (preferably having 1 carbon atom). -3 alkoxy group, more preferably methoxy group), hydroxyl group, aryl group (preferably phenyl group), alkyl group (preferably alkyl group having 1 to 5 carbon atoms, more preferably ethyl group, tert-butyl group), Acetyl group, N-alkyl-acetoamino group (the carbon number of alkyl is preferably 1 to 5), azide group and the like can be mentioned.
Among them, the N-alkyl-acetoamino group (more preferably N-methyl-acetoamino) is preferable in terms of antibacterial activity, because the nitrogen atom has strong intermolecular force and causes strong interaction with enzymes and cell membranes. Group, N-butyl-acetamino group) and azide group are preferable. In addition, an alkoxy group and a hydroxyl group are preferable because the interaction with a filler such as silica is strong.

Examples of allylic diphosphate (allylic diphosphate derivative) in which a portion of the isoprene unit is modified include compounds represented by the following formulas (A) to (S). Among them, since the interaction with the filler such as silica is stronger, low heat build-up, wet grip performance, wear resistance, elongation at break, and improvement in breaking strength are high, the following formulas (B), ( C), (D), (E), (F), (K), (L) and (R) are preferable, and the following formulas (B) and (C) are more preferable. Moreover, the structure represented by following formula (G)-(Q) is preferable, and the structure represented by following formula (K), (L), or (Q) is more preferable from the reason of being excellent in antimicrobial property.

Examples of allylic diphosphate derivatives represented by the above formulas (A) to (S) include dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranyl geranyl diphosphate, geraniol, farnesol, geranyl, etc. A person skilled in the art can manufacture it from Geraniol et al. With reference to the method described in JP-A-2012-036360.

（式（Ｙ）中、Ｒはメチル基以外の基を表す。） Next, the compound (R-IPP) represented by the following formula (Y) will be described.

(In formula (Y), R represents a group other than a methyl group.)

In the formula (Y), R is not particularly limited as long as it is a group other than a methyl group, but a nitrogen atom containing group, an oxygen atom containing group, a sulfur atom containing group, a silicon atom containing group, a carbon atom containing group Specifically, acetoxy, alkoxy, hydroxyl, aryl, alkyl (excluding methyl), acetyl, N-acetyl-acetamino, azide, amino And mercapto groups. Among them, a nitrogen atom-containing group, an oxygen atom-containing group, a sulfur atom-containing group, a silicon atom-containing group, a carbon atom-containing group, because they have high heat build-up, wet grip performance, elongation at break, and high improvement in breaking strength. (However, methyl group is excluded) is preferable, alkyl group (especially, alkyl group having 2 to 6 carbon atoms), aryl group (especially, phenyl group), mercapto group, hydroxyl group, amino group is more preferable, mercapto group, More preferred is a hydroxyl group.

The compounds represented by the above formula (Y) can be produced by those skilled in the art, for example, with reference to the methods described in the examples.

In the isoprene oligomer of the present invention, the number of isoprene units added to the allylic diphosphate represented by the above-mentioned formula (X), which is the starting substrate, is preferably 1 to 30, more preferably 1 to 20, still more preferably 1 To 10.

（式（Ｚ−１）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。）

（式（Ｚ−２）中、ｎは１〜１０の整数を表す。ｍは１〜３０の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。） Specific examples of the isoprene oligomer of the present invention include isoprene oligomers represented by the following formula (Z-1) or the following formula (Z-2), and the following formula (Z-1) or the following formula (Z) -2) is an isoprene oligomer in which at least one of the atoms or atomic groups contained in the v moiety is substituted by another atom or atomic group.

(In the formula (Z-1), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

(In the formula (Z-2), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxy carboxy group, an alkoxy carbonyl group or an OPP group Represents

The atoms or atomic groups contained in the v moiety in the above formula (Z-1) and the above formula (Z-2), and the other atoms or atomic groups are the same as those described for the above formula (X-1) The thing is mentioned.

The iv part in the said Formula (Z-1) and said Formula (Z-2) is a structure derived from the allylic diphosphate represented by said Formula (X) which is a starting substrate. Therefore, n in the said Formula (Z-1) and said Formula (Z-2) is the same as n in Formula (X).

The v moiety in the above formula (Z-1) and the above formula (Z-2) is an isoprene unit added to the allylic diphosphate represented by the above formula (X) which is the starting substrate. Therefore, m in the above formula (Z-1) and the above formula (Z-2) corresponds to the number of isoprene units added to the allylic diphosphate represented by the above formula (X) which is the above starting substrate Do.

As Y in the above formula (Z-1) and the above formula (Z-2), an excellent antimicrobial property is exhibited, and an interaction with a filler such as silica is strong. Groups are preferred.

（式（Ｚ−１−１）中のｎ、ｍ、Ｙは、式（Ｚ−１）中のｎ、ｍ、Ｙと同一である。式（Ｚ−１−１）中のＲは、式（Ｙ）中のＲと同一である。）

（式（Ｚ−２−１）中のｎ、ｍ、Ｙは、式（Ｚ−２）中のｎ、ｍ、Ｙと同一である。式（Ｚ−２−１）中のＲは、式（Ｙ）中のＲと同一である。） As described above, according to the present invention, even if the methyl group at the 3-position is substituted with a desired group by maintaining the structure of the moiety other than the methyl group at the 3-position of isopentenyl diphosphate. The present invention is based on the finding that isoprene oligomers and polyisoprene can be produced by using a naturally occurring enzyme. Therefore, the isoprene oligomer represented by said Formula (Z-1) or said Formula (Z-2) is an isoprene oligomer represented by a following formula (Z-1-1) or a following formula (Z-2-1). Is preferred.

(N, m and Y in the formula (Z-1-1) are the same as n, m and Y in the formula (Z-1). R in the formula (Z-1-1) is a formula (Y) identical to R in

(N, m and Y in the formula (Z-2-1) are the same as n, m and Y in the formula (Z-2). R in the formula (Z-2-1) is a formula (Y) identical to R in

Further, as described above, since it is preferable that the terminal part is also modified in addition to the main chain part, the above formula (Z-1) or the above formula (Z-2) (preferably the above formula ( It is preferable that at least one atom or atomic group contained in the iv moiety in Z-1-1) or the above formula (Z-2-1)) is substituted by another atom or atomic group.

Atom or atomic group contained in the iv part in the above formula (Z-1), above formula (Z-2), above formula (Z-1-1), above formula (Z-2-1), other atoms As the atomic group, the same ones as described for the above formula (X-1) can be mentioned.

Furthermore, as described above, the allylic diphosphate represented by the above formula (X) is an allylic diphosphate represented by the above formula (X-1), and in the above formula (X-1) At least one of the atoms or atomic groups contained in part II of the group is replaced by another atom or atomic group, and the atoms or atomic groups contained in part III in the above formula (X-1) are replaced by other atoms or atomic groups Preferably it is not substituted. Therefore, in the following formula (Z), the iv part in the above formula (Z-1), the above formula (Z-2), the above formula (Z-1-1) and the above formula (Z-2-1) is shown In the above formula (Z-1), the above formula (Z-2), the above formula (Z-1-1) and the above formula (Z-2-1), the iv part is in the following formula (Z) At least one of the atoms or atomic groups contained in the iv-II moiety is substituted by another atom or atomic group, and the atoms or atomic groups included in the iv-III moiety in the following formula (Z) are other atoms or atomic groups Preferably it is not substituted by

Examples of the atom or atomic group contained in the iv-II portion and the iv-III portion in the above formula (Z), and the other atoms or atomic groups include the same as those described for the above formula (X-1) Be

As a specific example of the iv portion when the terminal portion is also modified, for example, structures represented by the following formulas (a) to (s) can be mentioned. Among them, since the interaction with the filler such as silica is stronger, low heat build-up, wet grip performance, wear resistance, elongation at break, and improvement in breaking strength are high, the following formulas (b) and (b) c) The structures represented by (d), (e), (f), (k), (l) and (r) are preferable, and the structures represented by the following formulas (b) and (c) are more preferable preferable. Moreover, the structure represented by following formula (g)-(q) is preferable, and the structure represented by following formula (k), (l), or (q) is more preferable from the reason of being excellent in antimicrobial property.

(Method for producing isoprene oligomer)
The isoprene oligomer of the present invention is obtained by synthesis (biosynthesis) from allylic diphosphate represented by the above formula (X) and a compound represented by the above formula (Y).

As a method of synthesizing (biosynthesizing) the isoprene oligomer of the present invention from allylic diphosphate represented by the above formula (X) and the compound represented by the above formula (Y), for example, prenyltransferase activity The method of carrying out using an enzyme is included. Specifically, it has prenyl transferase activity with allylic diphosphate represented by the above formula (X) and a compound represented by the above formula (Y) which is a monomer to be polymerized to the allylic diphosphate The reaction may be performed in the presence of an enzyme. As the allylic diphosphate represented by the above formula (X) and the compound represented by the above formula (Y), only the same compound may be used, but a plurality of compounds may be used. It is also good. For example, an isoprene oligomer having desired properties can be obtained by using a plurality of compounds having different R in the above formula (Y) in combination as the compound represented by the above formula (Y).

In the present invention, only the compound (R-IPP) represented by the above formula (Y) may be used as a monomer to be polymerized to the allylic diphosphate represented by the above formula (X). In this case, an isoprene oligomer like the above-mentioned scheme 2 is obtained. Further, as a monomer to be polymerized to allylic diphosphate represented by the above formula (X), isopentenyl diphosphate (IPP) is used together with the compound (R-IPP) represented by the above formula (Y) May be In this case, an isoprene oligomer like the above-mentioned scheme 3 is obtained.

In the present specification, an enzyme having prenyltransferase activity is a new enzyme in which a condensation reaction between an allylic substrate (allylic diphosphate) and isopentenyl diphosphate is catalyzed and the isoprene unit is increased by 1 unit. By synthesizing allylic diphosphate, it means an enzyme having an activity of catalyzing a reaction of sequentially linking isopentenyl diphosphate to an allylic substrate (allylic diphosphate).
As described above, a compound represented by the above formula (Y) in which the methyl group at 3-position is substituted with a desired group while maintaining the structure of the moiety other than the 3-position methyl group of isopentenyl diphosphate In the case of -IPP), an isoprene oligomer can be produced by using a naturally occurring enzyme having prenyltransferase activity or the like.

The enzyme having the above prenyltransferase activity has already been confirmed in large numbers.
As an enzyme which extends a prenyl chain to Z type (a newly-made isoprene unit has a cis structure), for example, Z-nonaprenyl diphosphate synthetase (Ishii, K. et al., (1986) Biochem, J., 233, 773.), undecaprenyl diphosphate synthetase (Takahashi, I. and Ogura, K. (1982) J. Biochem., 92, 1527; Keenman, M. V. and Allen, C. M. (1974) Arch. Biochem. Biophys., 161, 375., Z-farnesyl diphosphate synthetase (Identification of a short (C-15) chain) Z-isoprenyl diphosphate synthase and a homologous long (C-50) chain isopr J. YL diphosphate synthetase in Mycobacterium tuberculosis, Schulbach, MC., et al. Shota. Et al, Biochimica Et Biophysica Acta (BBA), 1625 (3), (2003) p. 291-295.
Furthermore, examples of enzymes that cause the prenyl chain to extend to form E (a newly formed isoprene unit has a trans structure) include farnesyl diphosphate synthetase, geranylgeranyl diphosphate synthetase, hexaprenyl diphosphate synthetase, heptaprenyl Diphosphate synthetase, octaprenyl diphosphate synthetase, decaprenyl diphosphate synthetase and the like can be mentioned.
Since the maximum number of isoprene units that can be generated by each enzyme and the direction of extension of the prenyl chain (trans structure and cis structure) are determined, the enzyme used is changed according to the number of isoprene units and the direction of extension of the prenyl chain. do it. In the present invention, the extension direction (trans structure, cis structure) of the prenyl chain of the isoprene oligomer is not particularly limited. That is, the isoprene oligomer of the present invention may be, for example, an isoprene oligomer (for example, an isoprene oligomer represented by the above formula (Z-1)) in which all isoprene units are bonded in a trans form. The isoprene oligomer may be a cis-linked isoprene oligomer (for example, an isoprene oligomer represented by the above formula (Z-2)), or an isoprene oligomer having an isoprene unit bonded in a trans-cis-trans type, etc. .

Examples of organisms having the above enzyme having prenyltransferase activity but having all enzymes present on the earth but having the above prenyltransferase activity include, for example, Micrococcus luteus B-P26 (Microccus luteus B-P26), Escherichia coli (Escherichia coli), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Arabidopsis thaliana (Arabidopsis thaliana), Hevea braziliensis (Hevea brasiliensis), Periploca sepium (Periploca sepium) Stearothermophilu s) Sulfolobus acidocaldarius (Sulfolobus acidocaldarius, ATCC 49426), human (Homo sapiens), Nogeshi (Sonchus oleracers L.), dandelion (Taraxacum officinale), sunflower (Helianthus annuus) and the like.

The original substrate (starting substrate) of the enzyme having prenyltransferase activity is allylic diphosphate. And, since the allylic diphosphate derivative originally functions as an inhibitor of the enzyme, when the allylic diphosphate derivative is used as a starting substrate, in the enzyme, the allylic diphosphate is used. In many cases, the enzyme activity for acid derivatives (in particular, compounds represented by the above formulas (G) to (Q)) is low. Therefore, when using the said allylic diphosphate derivative as a starting substrate, you may use the mutant-type enzyme which improved the enzyme activity with respect to the said allylic diphosphate derivative. When a mutant-type enzyme is used, a living organism (transformant) transformed to express the mutant-type enzyme may be prepared by a genetic engineering method. Those skilled in the art can easily produce a mutant-type enzyme having improved enzyme activity with respect to allylic diphosphate derivatives by referring to the method described in the 036360 publication.

The isoprene oligomer of the present invention is obtained by reacting allylic diphosphate represented by the above formula (X) with a compound represented by the above formula (Y) in the presence of an enzyme having prenyl transferase activity. Be The presence of an enzyme having prenyltransferase activity refers to a culture of the above organism, an organism isolated from the culture, a treated product of the organism, an enzyme purified from the culture or the organism, genetic engineering method A culture of an organism (transformant) transformed to express an enzyme having prenyltransferase activity by the above, an organism separated from the culture, a treated product of the organism, the culture or the organism It means the situation where the enzyme etc. which were purified from.
An organism transformed to express an enzyme having prenyltransferase activity can be prepared by those skilled in the art according to conventionally known genetic engineering techniques.

In order to obtain an organism of the above-mentioned organism, the organism may be cultured in an appropriate medium. The medium for this purpose is not particularly limited as long as the organism can grow, and may be a common medium containing a usual carbon source, a nitrogen source, an inorganic ion, and, if necessary, an organic nutrient source.

For example, as the carbon source, any of the above-mentioned organisms can be used, and specifically, saccharides such as glucose, fructose, maltose, amylose and sucrose, alcohols such as sorbitol, ethanol and glycerol, fumaric acid, Organic acids such as citric acid, acetic acid, propionic acid and the like, salts thereof, carbohydrates such as paraffin or mixtures thereof can be used.

Nitrogen sources include ammonium salts of inorganic salts such as ammonium sulfate and ammonium chloride, ammonium salts of organic acids such as ammonium fumarate and ammonium citrate, nitrates such as sodium nitrate and potassium nitrate, peptone, yeast extract, meat extract, corn steep Organic nitrogen compounds such as liquor or mixtures thereof can be used.

In addition, nutrients such as inorganic salts, trace metal salts, vitamins, and hormones, which are used in a common medium, can be appropriately mixed and used.

The culture conditions are also not particularly limited, and, for example, the culture may be carried out for about 12 to 480 hours while appropriately limiting pH and temperature in the range of pH 5 to 8 and temperature 10 to 60 ° C. under aerobic conditions. .

The culture of the above-mentioned organism means, for example, a culture solution obtained by culturing the above-mentioned organism under the above-mentioned culture conditions, a culture filtrate (culture supernatant) obtained by separating an organism (organism) from the culture by filtration etc. Can be mentioned. Further, examples of the organism separated from the above culture include organisms (organisms) separated from the culture solution by filtration, centrifugation and the like.

Examples of the treated products of the above-mentioned organisms include crushed organisms obtained by homogenizing organisms separated from the above culture, and crushed organisms obtained by sonication.

The above-mentioned culture product or an enzyme purified from the above-mentioned organism means, for example, known purification procedures such as salting out the above-mentioned culture product or the enzyme present in the above-mentioned organism, ion exchange chromatography, affinity chromatography, gel filtration chromatography and the like It is an enzyme obtained by performing The purity of the purified enzyme is not particularly limited.

The isoprene oligomer of the present invention is obtained by reacting allylic diphosphate represented by the above formula (X) with a compound represented by the above formula (Y) in the presence of an enzyme having prenyl transferase activity. Specifically, for example, the culture or purification of the organism in a solution containing allylic diphosphate represented by the above formula (X) and a compound represented by the above formula (Y) The reaction may be carried out by adding an enzyme or the like. The reaction temperature may be, for example, 20 to 60 ° C., the reaction time may be, for example, 1 to 16 hours, and the pH may be, for example, 5 to 8. Moreover, you may add magnesium chloride, surfactant, 2-mercaptoethanol etc. as needed.

The isoprene oligomer of the present invention obtained by the above-mentioned reaction generally has the above-mentioned formula (Z-1), the above-mentioned formula (Z-2), the above-mentioned formula (Z-1-1) and the above-mentioned formula (Z-2-1) Y is an OPP group or a hydroxyl group. The OPP group is an OPP group derived from IPP or R-IPP. In addition, the OPP group is easily hydrolyzed, and the hydroxyl group is generated by the hydrolysis of the OPP group. Therefore, Y in the above formula (Z-1), the above formula (Z-2), the above formula (Z-1-1) and the above formula (Z-2-1) is usually an OPP group or a hydroxyl group .
In the isoprene oligomer in which Y in the formula (Z-1) and the formula (Z-2) is a formyl group, for example, Y in the formula (Z-1) and the formula (Z-2) is It is obtained by oxidizing an isoprene oligomer which is an OPP group.
In the isoprene oligomer in which Y in the above formula (Z-1) and the above formula (Z-2) is a carboxy group, for example, Y in the above formula (Z-1) and the above formula (Z-2) is It is obtained by oxidizing an isoprene oligomer which is an OPP group.
Moreover, the isoprene oligomer whose Y is an alkoxy carboxy group in the said Formula (Z-1) and said Formula (Z-2), and said Y in said Formula (Z-1) and said Formula (Z-2) is alkoxy The isoprene oligomer which is a carbonyl group is obtained, for example, by carboxylating and further esterifying an isoprene oligomer in which Y in the above formula (Z-1) and the above formula (Z-2) is an OPP group by the above method. Be

Since the isoprene oligomer of the present invention is obtained by biosynthesis except for organically synthesizing the compound represented by the above formula (Y) and the above allylic diphosphate derivative which is optionally a starting substrate, depletion of petroleum resources And environmental issues.

(Polyisoprene)
Next, the polyisoprene of the present invention will be described. The polyisoprene of the present invention is a polyisoprene obtained by synthesis (biosynthesis) from allylic diphosphate represented by the above formula (X) and a compound represented by the above formula (Y). In addition, as a monomer to be polymerized to allylic diphosphate represented by the above formula (X), isopentenyl diphosphate may be used together with the compound represented by the above formula (Y). For example, the polyisoprene of the present invention is obtained by being synthesized (biosynthesized) from the isoprene oligomer of the present invention, and the compound represented by the above formula (Y) and / or isopentenyl diphosphate. Moreover, the polyisoprene of the present invention can be obtained, for example, from an unmodified isoprene oligomer and a compound represented by the above formula (Y), or an unmodified isoprene oligomer, a compound represented by the above formula (Y), and iso It is obtained by synthesis (biosynthesis) from pentenyl diphosphate.

The polyisoprene of the present invention has a structure close to that of natural rubber and has high compatibility with rubber molecules. In addition, the polyisoprene of the present invention has a strong interaction with a filler such as silica because the main chain portion of the molecule is modified. Thus, the polyisoprene of the present invention is highly compatible with the rubber, and further, has a strong interaction with the filler such as silica. A rubber composition in which a molecule and a filler are combined can be obtained, and for example, the low heat buildup, wet grip performance, abrasion resistance, elongation at break, and breaking strength of the rubber composition can be improved.

In the polyisoprene of the present invention, polar groups and the like are present in the main chain portion. Therefore, the dispersibility of the filler such as silica is higher than in the case where a polar group (for example, a diphosphate group present at the end) is present only at the end, for example, low heat buildup, wet grip performance, Abrasion resistance, elongation at break, improvement in breaking strength are high.

Moreover, it is allylic diphosphate represented by said Formula (X-1) as allylic diphosphate represented by said Formula (X), Comprising: In II part in said Formula (X-1), At least one of the contained atoms or atomic groups is substituted by another atom or atomic group, and the atom or atomic group contained in the portion III in the above formula (X-1) is not substituted by another atom or atomic group When a compound is used, in addition to the main chain portion, polyisoprene in which the terminal portion is also modified is obtained. In addition to the main chain portion, since the end portion of the polyisoprene is modified, the interaction with the filler such as silica is further improved, and by incorporating it into the rubber composition, it is possible to more than before A rubber composition in which rubber molecules and a filler are combined in higher dimensions can be obtained, and for example, the low heat buildup, wet grip performance, abrasion resistance, elongation at break, and breaking strength of the rubber composition can be further improved.

In the polyisoprene of the present invention, the number of isoprene units added to the allylic diphosphate represented by the above formula (X), which is the starting substrate, is preferably 30 to 40,000, more preferably 31 to 38,000, still more preferably 1,000. It is preferably 35,000, particularly preferably 2,000 to 30,000.

（式（ＺＺ−１）中、ｎは１〜１０の整数を表す。ｑは３０〜４００００の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。）

（式（ＺＺ−２）中、ｎは１〜１０の整数を表す。ｑは３０〜４００００の整数を表す。Ｙは、水酸基、ホルミル基、カルボキシ基、アルコキシカルボキシ基、アルコキシカルボニル基又はＯＰＰ基を表す。） Specific examples of the polyisoprene of the present invention include polyisoprene represented by the following formula (ZZ-1) or the following formula (ZZ-2), and the following formula (ZZ-1) or the following formula (ZZ) At least one of the atoms or atomic groups contained in the v moiety in -2) is a polyisoprene substituted by another atom or atomic group.

(In the formula (ZZ-1), n represents an integer of 1 to 10. q represents an integer of 30 to 40. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

(In the formula (ZZ-2), n represents an integer of 1 to 10. q represents an integer of 30 to 40. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

The atom or atomic group contained in the v moiety in the above formula (ZZ-1) or the above formula (ZZ-2), and the other atoms or atomic groups are the same as those described for the above formula (X-1) The thing is mentioned.

The iv part in the said Formula (ZZ-1) and said Formula (ZZ-2) is a structure derived from the allylic diphosphate represented by said Formula (X) which is a starting substrate. Therefore, n in the formula (ZZ-1) and the formula (ZZ-2) is the same as n in the formula (X).

V part in said Formula (ZZ-1) and said Formula (ZZ-2) is an isoprene unit added to the allylic diphosphate represented by said Formula (X) which is a starting substrate. Therefore, q in the above formula (ZZ-1) and the above formula (ZZ-2) corresponds to the number of isoprene units added to the allylic diphosphate represented by the above formula (X) which is the above starting substrate Do.

As Y in the above formula (ZZ-1) and the above formula (ZZ-2), an OPP group, a hydroxyl group and a carboxy group are preferable because they have a strong interaction with a filler such as silica.

（式（ＺＺ−１−１）中のｎ、ｑ、Ｙは、式（ＺＺ−１）中のｎ、ｑ、Ｙと同一である。式（ＺＺ−１−１）中のＲは、式（Ｙ）中のＲと同一である。）

（式（ＺＺ−２−１）中のｎ、ｑ、Ｙは、式（ＺＺ−２）中のｎ、ｑ、Ｙと同一である。式（ＺＺ−２−１）中のＲは、式（Ｙ）中のＲと同一である。） As described above, according to the present invention, even if the methyl group at the 3-position is substituted with a desired group by maintaining the structure of the moiety other than the methyl group at the 3-position of isopentenyl diphosphate. The present invention is based on the finding that isoprene oligomers and polyisoprene can be produced by using a naturally occurring enzyme. Therefore, the polyisoprene represented by the said Formula (ZZ-1) or said Formula (ZZ-2) is a polyisoprene represented by a following formula (ZZ-1-1) or a following formula (ZZ-2-1). Is preferred.

(N, q and Y in the formula (ZZ-1-1) are the same as n, q and Y in the formula (ZZ-1). R in the formula (ZZ-1-1) is a formula (Y) identical to R in

(N, q and Y in the formula (ZZ-2-1) are the same as n, q and Y in the formula (ZZ-2). R in the formula (ZZ-2-1) is a formula (Y) identical to R in

Further, as described above, since it is preferable that the terminal part is also modified in addition to the main chain part, the above formula (ZZ-1) or the above formula (ZZ-2) (preferably the above formula ( It is preferable that at least one of the atoms or atomic groups contained in the iv moiety in ZZ-1-1) or the above formula (ZZ-2-1)) is substituted by another atom or atomic group.

Atom or atomic group contained in the iv moiety in the above formula (ZZ-1), above formula (ZZ-2), above formula (ZZ-1-1), above formula (ZZ-2-1), other atoms As the atomic group, the same ones as described for the above formula (X-1) can be mentioned.

Furthermore, as described above, the allylic diphosphate represented by the above formula (X) is an allylic diphosphate represented by the above formula (X-1), and in the above formula (X-1) At least one of the atoms or atomic groups contained in part II of the group is replaced by another atom or atomic group, and the atoms or atomic groups contained in part III in the above formula (X-1) are replaced by other atoms or atomic groups Preferably it is not substituted. Therefore, in the following formula (Z), the iv part in the above formula (ZZ-1), the above formula (ZZ-2), the above formula (ZZ-1-1) and the above formula (ZZ-2-1) is shown However, the iv part in said Formula (ZZ-1), said Formula (ZZ-2), said Formula (ZZ-1-1), and said Formula (ZZ-2-1) is in the following Formula (Z) At least one of the atoms or atomic groups contained in the iv-II moiety is substituted by another atom or atomic group, and the atoms or atomic groups included in the iv-III moiety in the following formula (Z) are other atoms or atomic groups Preferably it is not substituted by

Examples of the atom or atomic group contained in the iv-II portion and the iv-III portion in the above formula (Z), and the other atoms or atomic groups include the same as those described for the above formula (X-1) Be

As a specific example of the iv portion when the terminal portion is also modified, for example, structures represented by the above formulas (a) to (s) can be mentioned. Among them, the above formulas (b) and (b) are more effective because the interaction with the filler such as silica is stronger and the effect of improving the low heat buildup, wet grip performance, wear resistance, elongation at break, and break strength is high. The structures represented by c), (d), (e), (f), (k), (l) and (r) are preferable, and the structures represented by the above formulas (b) and (c) are more preferable. preferable.

(Method of producing polyisoprene)
As a method for producing the polyisoprene of the present invention, it is synthesized from (A) the isoprene oligomer of the present invention and the compound (R-IPP) represented by the above formula (Y) and / or isopentenyl diphosphate (IPP) (Biosynthesis) can be mentioned. The use ratio of the monomers R-IPP and IPP may be appropriately changed according to the target physical properties of the polyisoprene to be obtained, and one use ratio may be zero. Moreover, as a method for producing the polyisoprene of the present invention, (B) unmodified isoprene oligomer and a compound represented by the above formula (Y), or (C) unmodified isoprene oligomer, the above formula (Y) And compounds synthesized from isopentenyl diphosphate (biosynthesis). Also in this case, the use ratio of the monomers R-IPP and IPP may be appropriately changed in accordance with the target physical properties of the obtained polyisoprene.

Since the polyisoprene of the present invention is obtained by biosynthesis except for organically synthesizing the compound represented by the above formula (Y) and the above allylic diphosphate derivative which is optionally a starting substrate, the depletion of petroleum resources And environmental issues.

Conventionally, in natural rubber latex (especially, natural rubber latex derived from Hevea brasiliensis), the condensation reaction between isoprene oligomer and isopentenyl diphosphate is catalyzed, and isopeptenyl diphosphate is sequentially added to isoprene oligomer as Z type ( An enzyme having an activity of catalyzing the following reaction of forming a polyisoprene by linking newly formed isoprene units to a cis structure, or an enzyme which adds and polymerizes the rubber elongation factor (the above-mentioned isopentenyl diphosphate) Etc. are known to be included.

In addition, depending on the plant, a natural rubber latex (rubber latex) obtained from the plant is catalyzed a condensation reaction between an isoprene oligomer and isopentenyl diphosphate, and the isoprene oligomer is sequentially formed of isopentenyl diphosphate as an E-type (An enzyme having an activity of catalyzing a reaction which forms polyisoprene by linking a newly increased isoprene unit to a trans structure), a rubber elongation factor (such as an enzyme which adds and polymerizes the above-mentioned isopentenyl diphosphate), etc. It is also known that it contains.

As described above, a compound represented by the above formula (Y) in which the methyl group at 3-position is substituted with a desired group while maintaining the structure of the moiety other than the 3-position methyl group of isopentenyl diphosphate In the case of -IPP), polyisoprene can be produced by using an enzyme having an activity of catalyzing the above reaction for producing naturally occurring polyisoprene, a rubber elongation factor, and the like. Thus, in the present invention, polyisoprene can be produced using this enzyme, rubber elongation factor and the like.
That is, a method for synthesizing (biosynthesizing) the polyisoprene of the present invention from (A) the isoprene oligomer of the present invention and the compound represented by the above formula (Y) and / or isopentenyl diphosphate; A method for synthesizing (biosynthesizing) the polyisoprene of the present invention from a modified isoprene oligomer and a compound represented by the above formula (Y), or (C) an unmodified isoprene oligomer represented by the above formula (Y) Examples of the method for synthesizing (biosynthesizing) the polyisoprene of the present invention from the compound and isopentenyl diphosphate include, for example, methods using an enzyme contained in a natural rubber latex, a rubber elongation factor, and the like. Alternatively, an enzyme cloned from a natural rubber latex, a rubber elongation factor, or the like may be used.

That is, (A) the isoprene oligomer of the present invention may be reacted with the compound represented by the above formula (Y) and / or isopentenyl diphosphate in the presence of the above enzyme and / or the above rubber elongation factor . In addition, (B) the unmodified isoprene oligomer and the compound represented by the above formula (Y) may be reacted in the presence of the above enzyme and / or the above rubber elongation factor. In addition, (C) unmodified isoprene oligomer, a compound represented by the above formula (Y), and isopentenyl diphosphate may be reacted in the presence of the above enzyme and / or the above rubber elongation factor.
Specifically, for example, an enzyme separated from natural rubber latex or natural rubber latex in a solution containing the isoprene oligomer of the present invention, the compound represented by the above formula (Y) and / or isopentenyl diphosphate The reaction may be carried out by adding a rubber elongation factor or the like. The reaction temperature may be, for example, 10 to 60 ° C., the reaction time may be, for example, 1 to 72 hours, and the pH may be, for example, 6 to 8. Moreover, you may add magnesium chloride, surfactant, 2-mercaptoethanol, potassium fluoride etc. as needed. When using unmodified isoprene oligomer, the reaction may be carried out under the same conditions.

In the present invention, the extension direction (trans structure, cis structure) of the prenyl chain of polyisoprene is not particularly limited. That is, the polyisoprene of the present invention may be, for example, a polyisoprene in which all isoprene units are bound in trans form (for example, a polyisoprene represented by the above formula (ZZ-1)). It may be a polyisoprene having a cis bond (for example, a polyisoprene represented by the above formula (ZZ-2)), or may be a polyisoprene having an isoprene unit bonded to a trans-cis-trans form, etc. . Among them, polyisoprene in which an isoprene unit having a structure similar to that of a natural rubber derived from Hevea brasiliensis, which is widely used industrially, is bonded in a trans-cis type is preferable.

The polyisoprene of the present invention obtained by the above reaction generally has the above-mentioned formula (ZZ-1), the above-mentioned formula (ZZ-2), the above-mentioned formula (ZZ-1-1) and the above-mentioned formula (ZZ-2-1) Y is an OPP group or a hydroxyl group. The OPP group is an OPP group derived from IPP or R-IPP. In addition, the OPP group is easily hydrolyzed, and the hydroxyl group is generated by the hydrolysis of the OPP group. Therefore, Y in the above formula (ZZ-1), the above formula (ZZ-2), the above formula (ZZ-1-1) and the above formula (ZZ-2-1) is usually an OPP group or a hydroxyl group .
In the polyisoprene in which Y in the above formula (ZZ-1) and the above formula (ZZ-2) is a formyl group, for example, Y in the above formula (ZZ-1) and the above formula (ZZ-2) is It is obtained by oxidizing the polyisoprene which is an OPP group.
Further, in the polyisoprene in which Y in the above formula (ZZ-1) and the above formula (ZZ-2) is a carboxy group, for example, Y in the above formula (ZZ-1) and the above formula (ZZ-2) is It is obtained by oxidizing the polyisoprene which is an OPP group.
Moreover, polyisoprene in which Y in the above formula (ZZ-1) and above formula (ZZ-2) is an alkoxy carboxy group, and Y in the above formula (ZZ-1) and above formula (ZZ-2) is an alkoxy Polyisoprene which is a carbonyl group is obtained, for example, by carboxylating polyisoprene in which Y in the above formula (ZZ-1) and above formula (ZZ-2) is an OPP group by the above method and further esterifying it. Be

The origin of the natural rubber latex is not particularly limited. For example, Hevea brasiliensis, Ficus elastica, Ficus lyrata, Benjamin gum (Ficus benjamina), Indo borage (Ficus religiosa), Bengal borage (Ficus benghalensis), Chichitake (Lactarius volemus), Nogeshi (Sonchus oleracers L.), Dandelion (Taraxacum officinale), sunflower (Helianthus annuus) etc. are mentioned. Among them, Hevea brasiliensis is preferable because the molecular weight of the produced rubber is large and the amount of rubber contained in the latex is large.

The natural rubber latex is obtained, for example, by scratching the stem of Hevea brasiliensis in a groove shape with a knife or the like (tapping) and recovering the natural rubber latex flowing out of the cut milk duct.

Enzymes separated from natural rubber latex, rubber elongation factors, for example, are serum (Serum), liquid bottom phase (bottom fraction), rubber phase (rubber fraction), etc. which are separated by centrifuging natural rubber latex. It can be mentioned. The above-described enzymes, the above-mentioned rubber elongation factor, and the like are contained in the serum or liquid low phase or rubber phase.

(Rubber composition)
The rubber composition of the present invention comprises the isoprene oligomer of the present invention and / or the polyisoprene of the present invention. Therefore, the rubber composition of the present invention is excellent in low heat buildup, wet grip performance, wear resistance, elongation at break, breaking strength (in particular, low heat buildup, wear resistance). The polyisoprene of the present invention can be used as a rubber component.

The content of the polyisoprene of the present invention is preferably 20% by mass or more, more preferably 40% by mass or more, still more preferably 60% by mass or more, and may be 100% by mass in 100% by mass of the rubber component. .

Examples of rubber components that can be used other than the polyisoprene of the present invention include isoprene rubber (IR), natural rubber (NR), butadiene rubber (BR), styrene butadiene rubber (SBR), styrene isoprene butadiene rubber (SIBR), chloroprene Mention may be made of diene rubbers such as rubber (CR) and acrylonitrile butadiene rubber (NBR). The rubber component may be used alone or in combination of two or more. Among them, NR, BR and SBR are preferable.

When the isoprene oligomer of the present invention is blended in a rubber composition, it is preferable to use NR as the rubber component because the compatibility with the isoprene oligomer is high. By combining the isoprene oligomer of the present invention with NR, the effect of blending the isoprene oligomer of the present invention is more suitably obtained.

When the isoprene oligomer of the present invention is blended in a rubber composition, the content of NR in 100% by mass of the rubber component is preferably 20% by mass or more, more preferably 40% by mass or more, still more preferably 60% by mass or more It may be 100% by mass.

The content of the isoprene oligomer of the present invention is preferably 1 part by mass or more, more preferably 2 parts by mass or more, with respect to 100 parts by mass of the rubber component. If the amount is less than 1 part by mass, the effect obtained by blending the isoprene oligomer may not be sufficiently obtained. The content of the isoprene oligomer is preferably 20 parts by mass or less, more preferably 15 parts by mass or less. If it exceeds 20 parts by mass, the strength may be reduced, and the abrasion resistance may also be reduced.

As a filler which can be used by this invention, a silica, carbon black, clay, a calcium carbonate etc. are mentioned, for example.

In the present invention, it is preferred to use silica as the filler. By blending silica, the effects obtained by blending the isoprene oligomer of the present invention and / or the polyisoprene of the present invention are sufficiently obtained. The silica is not particularly limited, and examples thereof include dry method silica (anhydrous silicic acid), wet method silica (hydrous silicic acid) and the like, but wet method silica is preferable because it has many silanol groups.

In the present invention, it is also preferable to use carbon black as a filler. Also in this case, the effects obtained by blending the isoprene oligomer of the present invention and / or the polyisoprene of the present invention can be sufficiently obtained.

In the rubber composition of the present invention, in addition to the above-mentioned components, compounding agents generally used for producing a rubber composition, for example, a silane coupling agent, zinc oxide, stearic acid, various antioxidants, softening of oil and the like Agents, waxes, vulcanizing agents such as sulfur, vulcanization accelerators and the like can be appropriately blended.

As a method for producing the rubber composition of the present invention, a known method can be used. For example, the above-mentioned components are kneaded using a rubber kneading apparatus such as an open roll or a Banbury mixer and then vulcanized. It can be manufactured.

The rubber composition of the present invention can be suitably used for each component (for example, tread, sidewall, undertread, ply, breaker, carcass) of a tire.

(Pneumatic tire)
The pneumatic tire of the present invention can be produced by the usual method using the above rubber composition. That is, the rubber composition is extruded at the unvulcanized stage according to the shape of each member (for example, tread, sidewall) of the tire, and is molded by a usual method on a tire molding machine. It bonds together with a member and forms an unvulcanized tire. The unvulcanized tire can be heated and pressurized in a vulcanizer to produce a tire.

Although the present invention will be specifically described based on examples, the present invention is not limited to these.

(Production Example 1)
(Synthesis of 3-R-3-butyldiphosphate (compound represented by the above formula (Y) (R-IPP))
n-R-aldehyde was synthesized as a starting material. In n-R-aldehyde, according to the method of Green et al. (M. B. Green, and W. J. Hickinbotton, J. Chem. Soc., 1957, 3262), dimethylamine in formic acid, the exomethylene group at the alpha position Were introduced (the compound represented by (i) below). Next, reduction with lithium aluminum hydride was performed to obtain 2-R-allylalcohol (a compound represented by (ii) below). Furthermore, chlorination was carried out with phosphorus chloride in pyridine, and via 2-R-allyloride (a compound represented by (iii) below), a Grignard reagent was synthesized and carbon dioxide was reacted to be a carboxylic acid (following (iv A compound represented by). Next, it was reduced with lithium aluminum hydride to obtain an alcohol (compound represented by (v) below). Next, it was tosylated with tosyl chloride in pyridine (compound represented by (vi) below). Next, the product is phosphorylated with trimethyl phosphate in acetonitrile to obtain the desired product (the compound represented by the following (vii) (the compound represented by the above formula (Y) (R-IPP)) . Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).
In addition, the compound (R-IPP) represented with the said Formula (Y) was synthesize | combined about the case where said R is an ethyl group, a propyl group, a butyl group, a phenyl group, a mercapto group, a hydroxyl group, and an amino group. Further, R-IPP in which R in the above-mentioned formula (Y) is an ethyl group, a propyl group, a butyl group, a phenyl group, a mercapto group, a hydroxyl group or an amino group is referred to below as R-IPP-A, It also describes R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F, and R-IPP-G.

Example 1
(Preparation of isoprene oligomer (all-trans))
Preparation of the isoprene oligomer which the isoprene unit represented by a following formula (Z-1-1-1) couple | bonded with trans type was performed.

(Preparation of transformant)
First, transformants were prepared. For preparation of transformants, pET15b (pET15b / human-GGPS) into which human-derived geranylgeranyl diphosphate synthase had been incorporated was used. PET15b / human-GGPS was transferred from Associate Professor Hiroshi Sakami of Institute of Multi-Material Science, Tohoku University.
Heat shock method E. coli using pET15b / human-GGPS. E. coli BL21 (DE3) was transformed, and the transformant was coated on LB agar medium containing 50 μg / mL of ampicillin and cultured overnight at 37 ° C. to select transformants.

(Production of a protein having prenyltransferase activity)
Obtained E. coli. E. coli BL21 (DE3) / pET15b / human-GGPS (wild-type) was inoculated into a test tube containing 3 mL of LB medium containing 50 μg / mL of ampicillin, and shake cultured at 37 ° C. for 5 hours. Inoculate 1 mL of the obtained culture solution into a 500 mL Erlenmeyer flask containing 100 mL of LB medium containing 50 μg / mL of ampicillin, shake culture at 37 ° C. for 3 hours, and add IPTG to 0.1 mmol / L. And shake cultured at 30 ° C. for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells obtained above were disrupted by sonication, and then the supernatant obtained by centrifugation was purified using a HisTrap (manufactured by Amersham) using a protein having a prenyltransferase activity. The purified protein was confirmed to be purified by SDS-PAGE.

(Preparation of isoprene oligomer)
10 mg of purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 25 mM 2-mercaptoethanol, 1 mM starting substrate (geranyl diphosphate (GPP)), 1 mM isopentenyl diphosphate (IPP) or The reaction solution containing each R-IPP prepared in Preparation Example 1 was prepared, and reacted at 37 ° C. water bath for 1 hour. After completion of the reaction, 100 ml of saturated brine and 500 ml of pentane were added, and after stirring, the mixture was allowed to stand. Thereafter, the supernatant (pentane layer) was concentrated to dryness by evaporation. The structure was partially confirmed by NMR to obtain an isoprene oligomer. The details (n, m, R in the formula (Z-1-1-1)) of the obtained isoprene oligomer are shown in Table 1. In addition, n and m in a formula (Z-1-1-1) were computed based on the information of the used starting substrate, and the isoprene chain length by TLC. Moreover, R in Formula (Z-1-1-1) identified the structure by NMR, TLC, and GC-MS.
In addition, IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F, and R-IPP-G are used as monomers. The isoprene oligomer obtained by this method is respectively obtained from isoprene oligomer (t-control), isoprene oligomer (t-A), isoprene oligomer (t-B), isoprene oligomer (t-C), isoprene oligomer (t-D) , Isoprene oligomer (t-E), isoprene oligomer (t-F), and isoprene oligomer (t-G) were used in the following experiments. Moreover, isoprene oligomer (t-E) and isoprene oligomer (t-F) were mix | blended with the rubber composition by the following experiments as isoprene oligomer a and isoprene oligomer e.

(Relative activity of each monomer)
The reaction was carried out using each R-IPP prepared in IPP or Preparation Example 1 and geranyl diphosphate (GPP) under the following conditions, and the relative activity of each R-IPP against GPP was compared with the activity in the case of IPP. The index is displayed as 100.
500 ng of purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 25 mM 2-mercaptoethanol, 12.5 μM GPP, 50 μM [1- ¹⁴ C] IPP or each R-prepared in Preparation Example 1 The reaction solution containing IPP was adjusted, and reacted at 37 ° C. water bath for 1 hour. After the reaction, the activity under each condition was measured by quantifying the value of liquid scintillation and TLC, and the relative activity of each R-IPP was shown in Table 2 with the activity for IPP as 100.
In addition, isoprene oligomer was purified in the same manner as the above (Example 1) (preparation of isoprene oligomer). And the detail (n, m, R in a formula (Z-1-1-1)) of the obtained isoprene oligomer was shown in Table 2. In addition, n and m in a formula (Z-1-1-1) were computed based on the information of the used starting substrate, and the isoprene chain length by TLC. Moreover, R in Formula (Z-1-1-1) identified the structure by NMR, TLC, and GC-MS.

From the results of Tables 1 and 2, even when R-IPP was used, an isoprene oligomer having the same molecular weight as when IPP was used was obtained. In addition, it was also confirmed that the main chain of the obtained isoprene oligomer was modified according to the R-IPP used.

(Example 2)
(Preparation of polyisoprene)
Next, polyisoprene was prepared using an isoprene oligomer (all-trans) in which all isoprene units represented by the above formula (Z-1-1-1) were bonded in a trans form.
10 μl of the latex component, 50 mM Tris-HCl buffer (pH 7.5), 25 mM magnesium chloride, 40 mM 2-mercaptoethanol, 40 mM potassium fluoride, 50 μM isoprene oligomer, 1 mM IPP or each R-IPP prepared in Preparation Example 1 The reaction solution was adjusted, and reacted at 30 ° C. water bath for 3 days. After the reaction, the molecular weight was measured by GPC. Then, based on the measured molecular weight and information of the starting substrate used, the number of isoprene units added to the starting substrate GPP was calculated. The results are shown in Table 3.

As a latex component, a serum prepared by ultracentrifugation of a latex obtained from Hevea brasiliensis was used.

The isoprene oligomer is isoprene oligomer (t-control), isoprene oligomer (t-A), isoprene oligomer (t-B), isoprene oligomer (t-C), isoprene oligomer prepared by the above (preparation of isoprene oligomer) (T-D), isoprene oligomer (t-E), isoprene oligomer (t-F), isoprene oligomer (t-G) were used.

From the results in Table 3, even when R-IPP was used, polyisoprene having the same molecular weight as when IPP was used was obtained. Further, as a result of analyzing the obtained polyisoprene by NMR, TLC and GC-MS, it can be confirmed that the main chain of polyisoprene is modified according to the used R-IPP as in the case of isoprene oligomer. The

(Example 3)
(Preparation of isoprene oligomer (trans-cis))
Preparation of the isoprene oligomer which the isoprene unit represented by following formula (Z-2-1-1) couple | bonded in the trans-cis form was performed.

(Preparation of transformant)
First, transformants were prepared. For preparation of transformants, pET22b (pET22b / MLU-UPS) in which undecaprenyl diphosphate synthetase from Micrococcus luteus B-P26 was incorporated was used as a dsDNA template. In addition, pET22b / MLU-UPS was transferred from Prof. Terutoshi Tateyama, Research Institute for Advanced Materials Science, Tohoku University.
Heat shock method E. coli using pET 22b / MLU-UPS. E. coli BL21 (DE3) was transformed, and the transformant was coated on LB agar medium containing 50 μg / mL of ampicillin and cultured overnight at 37 ° C. to select transformants.

(Production of a protein having prenyltransferase activity)
Obtained E. coli. E. coli BL21 (DE3) / pET22b / MLU-UPS was inoculated into a test tube containing 3 mL of LB medium containing 50 μg / mL of ampicillin, and shake cultured at 37 ° C. for 5 hours. Inoculate 1 mL of the obtained culture solution into a 500 mL Erlenmeyer flask containing 100 mL of LB medium containing 50 μg / mL of ampicillin, shake culture at 37 ° C. for 3 hours, and add IPTG to 0.1 mmol / L. And shake cultured at 30 ° C. for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells obtained above were disrupted by sonication, and then the supernatant obtained by centrifugation was purified using a HisTrap (manufactured by Amersham) using a protein having a prenyltransferase activity. The purified protein was confirmed to be purified by SDS-PAGE.

(Preparation of isoprene oligomer)
10 mg of purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 40 mM Triton X-100, 25 mM 2-mercaptoethanol, 1 mM starting substrate (farnesyl diphosphate (FPP)), 1 mM IPP or The reaction solution containing each R-IPP prepared in Preparation Example 1 was prepared, and reacted at 37 ° C. water bath for 1 hour. After completion of the reaction, 100 ml of saturated saline and 500 ml of 1-butanol were added, and after stirring, the mixture was allowed to stand. Thereafter, the supernatant (1-butanol layer) was concentrated to dryness by evaporation. The structure was partially confirmed by NMR to obtain an isoprene oligomer. The details (n, m, R in formula (Z-2-1-1)) of the obtained isoprene oligomer are shown in Table 4. In the formula (Z-2-1-1), n and m were calculated based on the information on the starting substrate used and the isoprene chain length by TLC. Moreover, R in Formula (Z-2-1-1) identified the structure by NMR, TLC, and GC-MS.
As monomers, IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F, R-IPP-G, respectively Isoprene oligomers (tc-control), isoprene oligomers (tc-A), isoprene oligomers (tc-B), isoprene oligomers (tc-C), isoprene oligomers (tc-D) obtained by using ), Isoprene oligomer (tc-E), isoprene oligomer (tc-F), and isoprene oligomer (tc-G) in the following experiments. Moreover, isoprene oligomer (tc-E) was mix | blended with the rubber composition by the following experiments as isoprene oligomer b. Furthermore, isoprene oligomer (tc-A), isoprene oligomer (tc-B), isoprene oligomer (tc-C), isoprene oligomer (tc-D), isoprene oligomer (tc-E), isoprene oligomer (tc-F), An isoprene oligomer (tc-G) was blended into the rubber composition as an isoprene oligomer h, an isoprene oligomer i, an isoprene oligomer j, an isoprene oligomer k, an isoprene oligomer 1, an isoprene oligomer m, and an isoprene oligomer n in the following experiment.

(Relative activity of each monomer)
The reaction is carried out using IPP or each R-IPP prepared in Preparation Example 1 and farnesyl diphosphate (FPP) under the following conditions, and the relative activity of each R-IPP against FPP is compared with the activity in the case of IPP. The index is displayed as 100.
500 ng of purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 40 mM Triton X-100, 25 mM 2-mercaptoethanol, 12.5 μM FPP, 50 μM [1- ¹⁴ C] IPP or Preparation Example 1 The reaction liquid containing each prepared R-IPP was adjusted, and was made to react at 37 degreeC water bath for 1 hour. After the reaction, the activity under each condition was measured by quantifying the value of liquid scintillation and TLC, and the relative activity of each R-IPP was shown in Table 5 with the activity as IPP as 100.
Also, the isoprene oligomer was purified in the same manner as the above (Example 3) (preparation of isoprene oligomer). And the detail (n, m, R in a formula (Z-2-1-1)) of the obtained isoprene oligomer was shown in Table 5. In the formula (Z-2-1-1), n and m were calculated based on the information on the starting substrate used and the isoprene chain length by TLC. Moreover, R in Formula (Z-2-1-1) identified the structure by NMR, TLC, and GC-MS.

From the results of Tables 4 and 5, even when R-IPP was used, isoprene oligomers having the same molecular weight as when IPP was used were obtained. In addition, it was also confirmed that the main chain of the obtained isoprene oligomer was modified according to the R-IPP used.

(Example 4)
(Preparation of polyisoprene)
Next, preparation of polyisoprene was performed using the isoprene oligomer (trans-cis) which the isoprene unit represented by said Formula (Z-2-1-1) couple | bonded in the trans-cis form.
10 μl of the latex component, 50 mM Tris-HCl buffer (pH 7.5), 25 mM magnesium chloride, 40 mM 2-mercaptoethanol, 40 mM potassium fluoride, 50 μM isoprene oligomer, 1 mM IPP or each R-IPP prepared in Preparation Example 1 The reaction solution was adjusted, and reacted at 30 ° C. water bath for 3 days. After the reaction, the molecular weight was measured by GPC. Then, based on the measured molecular weight and the information on the used starting substrate, the number of isoprene units added to the starting substrate FPP was calculated. The results are shown in Table 6.

As a latex component, a serum prepared by ultracentrifugation of a latex obtained from Hevea brasiliensis was used.

The isoprene oligomer is isoprene oligomer (tc-control), isoprene oligomer (tc-A), isoprene oligomer (tc-B), isoprene oligomer (tc-C), isoprene oligomer prepared by the above (preparation of isoprene oligomer) (Tc-D), isoprene oligomer (tc-E), isoprene oligomer (tc-F), and isoprene oligomer (tc-G) were used. In addition, the polyisoprene obtained by using an isoprene oligomer (tc-E) and an isoprene oligomer (tc-F) was mix | blended with the rubber composition by the following experiments as polyisoprene D and polyisoprene A. Furthermore, isoprene oligomer (tc-A), isoprene oligomer (tc-B), isoprene oligomer (tc-C), isoprene oligomer (tc-D), isoprene oligomer (tc-E), isoprene oligomer (tc-F), As polyisoprene I, polyisoprene J, polyisoprene K, polyisoprene L, polyisoprene M, polyisoprene N, polyisoprene P, the following experiments were carried out using the polyisoprene I obtained using isoprene oligomer (tc-G) Were blended into the rubber composition.

From the results in Table 6, even when R-IPP was used, polyisoprene having the same molecular weight as when IPP was used was obtained. Further, as a result of analyzing the obtained polyisoprene by NMR, TLC and GC-MS, it can be confirmed that the main chain of polyisoprene is modified according to the used R-IPP as in the case of isoprene oligomer. The

(Example 5)
(Preparation of isoprene oligomer (all-trans))
Preparation of the isoprene oligomer which the isoprene unit represented by a following formula (Z-1-1-1) couple | bonded with trans type was performed.

(Preparation of transformant)
First, transformants were prepared. For preparation of transformants, pET15b (pET15b / bacillus-FPS) in which farnesyl diphosphate synthase derived from Bacillus Sterarothermophilus was incorporated was used. In addition, pET15b / bacillus-FPS was transferred from Prof. Terutoshi Koyama of Institute of Multidisciplinary Research for Materials Science, Tohoku University.
E. coli by heat shock method using pET15b / bacillus-FPS. E. coli BL21 (DE3) was transformed, and the transformant was coated on LB agar medium containing 50 μg / mL of ampicillin and cultured overnight at 37 ° C. to select transformants.

(Production of a protein having prenyltransferase activity)
Obtained E. coli. E. coli BL21 (DE3) / pET15b / bacillus-FPS (wild type) was inoculated into a test tube containing 3 mL of LB medium containing 50 μg / mL of ampicillin, and shake cultured at 37 ° C. for 5 hours. Inoculate 1 mL of the obtained culture solution into a 500 mL Erlenmeyer flask containing 100 mL of LB medium containing 50 μg / mL of ampicillin, shake culture at 37 ° C. for 3 hours, and add IPTG to 0.1 mmol / L. And shake cultured at 30 ° C. for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells obtained above were disrupted by sonication, and then the supernatant obtained by centrifugation was purified using a HisTrap (manufactured by Amersham) using a protein having a prenyltransferase activity. The purified protein was confirmed to be purified by SDS-PAGE.

(Preparation of isoprene oligomer)
10 mg of purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 25 mM 2-mercaptoethanol, 1 mM starting substrate (dimethylallyl diphosphate (DMAPP)), 1 mM isopentenyl diphosphate (IPP) Alternatively, a reaction solution containing each R-IPP prepared in Preparation Example 1 was prepared, and reacted at 37 ° C. water bath for 1 hour. After completion of the reaction, 100 ml of saturated brine and 500 ml of pentane were added, and after stirring, the mixture was allowed to stand. Thereafter, the supernatant (pentane layer) was concentrated to dryness by evaporation. The structure was partially confirmed by NMR to obtain an isoprene oligomer. The details (n, m, R in the formula (Z-1-1-1)) of the obtained isoprene oligomer are shown in Table 7. In addition, n and m in a formula (Z-1-1-1) were computed based on the information of the used starting substrate, and the isoprene chain length by TLC. Moreover, R in Formula (Z-1-1-1) identified the structure by NMR, TLC, and GC-MS.
In addition, IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F, and R-IPP-G are used as monomers. The isoprene oligomer obtained by this method is respectively obtained from isoprene oligomer (t-control-1), isoprene oligomer (t-A-1), isoprene oligomer (t-B-1), and isoprene oligomer (t-C-1). , Isoprene oligomer (t-D-1), isoprene oligomer (t-E-1), isoprene oligomer (t-F-1), and isoprene oligomer (t-G-1) in the following experiments.

(Relative activity of each monomer)
The reaction was carried out using each R-IPP prepared in IPP or Preparation Example 1 and dimethyl diphosphate (DMAPP) under the following conditions, and the relative activity of each R-IPP to DMAPP was compared to that of IPP. The index is displayed as 100.
500 ng of purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 25 mM 2-mercaptoethanol, 12.5 μM DMAPP, 50 μM [1- ¹⁴ C] IPP or each R prepared in Preparation Example 1 The reaction solution containing IPP was adjusted, and reacted at 37 ° C. water bath for 1 hour. After the reaction, the activity under each condition was measured by quantifying the value of liquid scintillation and TLC, and the relative activity of each R-IPP was shown in Table 8 with the activity as IPP as 100.
Also, the isoprene oligomer was purified in the same manner as the above (Example 5) (Preparation of isoprene oligomer). And the detail (n, m, R in a formula (Z-1-1-1)) of the obtained isoprene oligomer was shown in Table 8. In addition, n and m in a formula (Z-1-1-1) were computed based on the information of the used starting substrate, and the isoprene chain length by TLC. Moreover, R in Formula (Z-1-1-1) identified the structure by NMR, TLC, and GC-MS.

From the results of Tables 7 and 8, even when R-IPP was used, isoprene oligomers having the same molecular weight as when IPP was used were obtained. In addition, it was also confirmed that the main chain of the obtained isoprene oligomer was modified according to the R-IPP used.

(Example 6)
(Preparation of polyisoprene)
Next, polyisoprene was prepared using an isoprene oligomer (all-trans) in which all isoprene units represented by the above formula (Z-1-1-1) were bonded in a trans form.
10 μl of the latex component, 50 mM Tris-HCl buffer (pH 7.5), 25 mM magnesium chloride, 40 mM 2-mercaptoethanol, 40 mM potassium fluoride, 50 μM isoprene oligomer, 1 mM IPP or each R-IPP prepared in Preparation Example 1 The reaction solution was adjusted, and reacted at 30 ° C. water bath for 3 days. After the reaction, the molecular weight was measured by GPC. Then, based on the measured molecular weight and information on the used starting substrate, the number of isoprene units added to the starting substrate DMAPP was calculated. The results are shown in Table 9.

As a latex component, a serum prepared by ultracentrifugation of a latex obtained from Hevea brasiliensis was used.

In addition, isoprene oligomers are isoprene oligomers (t-control-1), isoprene oligomers (t-A-1), isoprene oligomers (t-B-1), isoprene oligomers (t) prepared by the above (preparation of isoprene oligomers) -C-1), isoprene oligomer (t-D-1), isoprene oligomer (t-E-1), isoprene oligomer (t-F-1), isoprene oligomer (t-G-1) were used.

From the results of Table 9, even when R-IPP was used, polyisoprene having the same molecular weight as when IPP was used was obtained. Further, as a result of analyzing the obtained polyisoprene by NMR, TLC and GC-MS, it can be confirmed that the main chain of polyisoprene is modified according to the used R-IPP as in the case of isoprene oligomer. The

(Example 7)
(Preparation of isoprene oligomer (trans-cis))
Next, using an allylic diphosphate derivative, it is an isoprene oligomer in which an isoprene unit represented by the following formula (Z-2-1-1) is bound in a trans-cis form, and is added to the main chain portion Then, an isoprene oligomer (main chain terminal modified isoprene oligomer) was also prepared in which the terminal portion was also modified.

(Preparation of allylic diphosphate derivative)
First, an allylic diphosphate derivative was synthesized.

(Production Example 2)
(Synthesis of 8-metoxy-3,7-dimetyl-dodeca- (2E, 6E) -dienyl diphosphate (compound represented by the above formula (B)))
Geraniol was synthesized as the starting material. Acetylation was carried out using anhydrous pyridine and acetic anhydride in anhydrous dichloromethane to obtain an acetate (compound represented by (bi) below) (yield: 95%). Next, selenium was oxidized to carbon at position 8 in ethanol to obtain an aldehyde (a compound represented by (bii) below) (yield: 24%). Next, alkaline hydrolysis was carried out using potassium hydroxide to obtain an alcohol form (compound represented by (biii) below) (yield: 38%). Then, imidazole, tert-butyl diphenyl silyl chloride (TBDPS) was used in anhydrous dichloromethane to obtain (compound represented by (biv) below) (yield: 80%). Thereafter, butyl lithium was reacted in anhydrous ether to obtain a butyl alcohol (compound represented by (bv) below) (yield: 73%). Next, after making it sodium salt with sodium hydroxide in anhydrous tetrahydrafuran, methyl iodide was added, and the ether form (compound represented by the following (bvi)) was obtained by Williamson synthesis (yield 95%). Next, it was eliminated using anhydrous tetrahydrafuran with tetra-n-ammonium fluoride to obtain an alcohol form (compound represented by (bvii) below) (yield 87%). Next, the primary hydroxyl group was subjected to chlorine substitution using N-chlorosuccinimide and dimethyl sulfide in an anhydrous dichloromethane solvent at -40 ° C. or lower to obtain a chloride (compound represented by (bviii) below) (yield 92) %). Next, it is diphosphorylated using tris-tetra n-butyl ammonium hydride phosphate in anhydrous acetonitrile, and the target substance (the compound represented by (bix) below (the compound represented by the above formula (B))) (Yield 26%).
Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Production Example 3)
(Synthesis of 8-hydroxy-3,7-dimetyl-dodeca- (2E, 6E) -dienyl diphosphate (compound represented by the above formula (C)))
Geraniol was synthesized as the starting material. Acetylation was carried out using anhydrous pyridine and acetic anhydride in anhydrous dichloromethane to obtain an acetate (compound represented by (ci) below) (yield: 97%). Next, selenium was oxidized to carbon at position 8 in ethanol to obtain an aldehyde (compound represented by (cii) below) (yield: 20%). Next, alkaline hydrolysis was performed using potassium hydroxide to obtain an alcohol form (compound represented by (ciii) below) (yield: 42%). Then, imidazole, tert-butyldiphenylsilyl chloride (TBDPS) was used in anhydrous dichloromethane to obtain (compound represented by (civ) below) (yield: 80%). Thereafter, butyllithium was reacted in anhydrous ether to obtain a butyl alcohol (yield 62%). Next, it was eliminated using anhydrous tetrahydrafuran with tetra-n-ammonium fluoride to obtain a diol (compound represented by (cvi) below) (yield: 94%). Next, the primary hydroxyl group was subjected to chlorine substitution using N-chlorosuccinimide and dimethyl sulfide in an anhydrous dichloromethane solvent or less at −40 ° C. or less to obtain a chloride (a compound represented by (cvii) below) (yield 90 %). Next, it is diphosphorylated using tris-tetra n-butyl ammonium hydride phosphate in anhydrous acetonitrile, and the target substance (the compound represented by (cviii) below (the compound represented by the above formula (C))) (Yield 46%).
Confirmation of intermediates and final products in each synthesis step was performed using TLC and instrumental analysis (IR, NMR).

(Preparation of mutagenizing enzyme)
When an allylic diphosphate derivative is used as a starting substrate, the reaction can be allowed to proceed even using an enzyme having wild-type prenyltransferase activity to prepare an isoprene oligomer. However, in order to improve the reaction efficiency, it is allylic. We produced mutant enzymes with improved enzyme activity for diphosphate derivatives.
The reagents used were Stratagene's QuickChange Site-Directed Mutagenesis Kit. The primers were designed to introduce a mutation at the target site. Primers for mutation introduction were purchased from Medical Biology Research Institute, Inc. (manufacturer: IDT). The designed primers are as shown below.
Primer sense primer for preparation of mutant type enzyme N77A 5'-act gaa gca tgg tct cgt cct aaa g-3 '(SEQ ID NO: 1)
Antisense primer 5'-gag acc atg ctt cag ttg aaa atg c-3 '(SEQ ID NO: 2)
Primer sense primer for preparation of mutant type enzyme L91D 5′-gaat aaa tcc ggg tga ttt ttt aa-3 ′ (SEQ ID NO: 3)
Antisense primer 5'-cac ccg gat ctt tca agt aat ta-3 '(SEQ ID NO: 4)

The dsDNA template used was pET22b (pET22b / MLU-UPS) into which undecaprenyl diphosphate synthetase derived from Micrococcus luteus B-P26 (hereinafter also referred to as a wild-type enzyme) was incorporated. In addition, pET22b / MLU-UPS was transferred from Prof. Furuyama Tanetoshi, Research Institute for Advanced Materials Science, Tohoku University. 2 μl of 10x Pfu polymerase buffer, 2-20 ng of dsDNA template, 50 ng of sense primer, 50 ng of antisense primer, 0.4 μl of 2.5 mM each dNTP, 20 μl of ddH ₂ O, 0.4 ml of Pfu polymerase (2.5 U / μl) Mix and perform PCR reaction. The PCR reaction was carried out at 95 ° C. for 30 sec in one cycle and at 95 ° C. for 30 sec-55 ° C. for 1 min-68 ° C. for 8 min for 15 cycles. After PCR, 0.4 μl of Dpn I was added to the PCR reaction solution, and Dpn I treatment was performed at 37 ° C. for 1 hour. Heat shock method E. coli using 1-10 μl of Dpn I treatment solution. E. coli DH5α was transformed, and the transformant was coated on LB agar medium containing 50 μg / mL of ampicillin and cultured overnight at 37 ° C. to select transformants. The transformant was cultured overnight in LB medium containing 50 μg / ml of ampicillin, and a plasmid was prepared from the obtained culture medium by the alkaline SDS method. The plasmid was confirmed to be mutagenized using a sequencer.

(Production of a protein having prenyltransferase activity)
Obtained E. coli. E. coli BL21 (DE3) / pET22b / MLU-UPS (wild type and mutant) were inoculated into a test tube containing 3 mL of LB medium containing 50 μg / mL of ampicillin, and shake cultured at 37 ° C. for 5 hours. Inoculate 1 mL of the obtained culture solution into a 500 mL Erlenmeyer flask containing 100 mL of LB medium containing 50 μg / mL of ampicillin, shake culture at 37 ° C. for 3 hours, and add IPTG to 0.1 mmol / L. And shake cultured at 30 ° C. for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells obtained above were disrupted by sonication, and then the supernatant obtained by centrifugation was purified using a HisTrap (manufactured by Amersham) using a protein having a prenyltransferase activity. The purified protein was confirmed to be purified by SDS-PAGE. The amino acid sequences of the resulting mutant enzyme N77A and mutant enzyme L91D are shown in SEQ ID NOS: 5 and 6 of the Sequence Listing.

(Preparation of isoprene oligomer)
10 mg of each purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 40 mM Triton X-100, 25 mM 2-mercaptoethanol, 1 mM starting substrate (farnesyl diphosphate (FPP) or Preparation Example 2 The reaction solution containing the allylic diphosphate derivative prepared in 3), 1 mM IPP, or each R-IPP prepared in Preparation Example 1 was prepared, and reacted at 37 ° C. water bath for 1 hour. After completion of the reaction, 100 ml of saturated saline and 500 ml of 1-butanol were added, and after stirring, the mixture was allowed to stand. Thereafter, the supernatant (1-butanol layer) was concentrated to dryness by evaporation. The structure was partially confirmed by NMR to obtain an isoprene oligomer. The details (n, m, R in the formula (Z-2-1-1)) of the obtained isoprene oligomer are shown in Table 10. In the formula (Z-2-1-1), n and m were calculated based on the information on the starting substrate used and the isoprene chain length by TLC. Moreover, R in Formula (Z-2-1-1) identified the structure by NMR, TLC, and GC-MS.
In addition, a mutant enzyme N77A as an enzyme, a compound represented by the above formula (B) as a starting substrate, and IPP, R-IPP-A, R-IPP-B, R-IPP-C, R- as a monomer are used. The isoprene oligomer obtained by using IPP-D, R-IPP-E, R-IPP-F, and R-IPP-G, respectively, was isoprene oligomer (tc-control-AB) and isoprene oligomer (tc-, respectively). A-AB), isoprene oligomer (tc-B-AB), isoprene oligomer (tc-C-AB), isoprene oligomer (tc-D-AB), isoprene oligomer (tc-E-AB), isoprene oligomer (tc- F-AB), used as an isoprene oligomer (tc-G-AB) in the following experiments.
In addition, a mutant enzyme L91D as an enzyme, a compound represented by the above formula (B) as a starting substrate, and IPP, R-IPP-A, R-IPP-B, R-IPP-C, R- as monomers are used. The isoprene oligomer obtained by using IPP-D, R-IPP-E, R-IPP-F, and R-IPP-G, respectively, is isoprene oligomer (tc-control-DB), isoprene oligomer (tc- A-DB), isoprene oligomer (tc-B-DB), isoprene oligomer (tc-C-DB), isoprene oligomer (tc-D-DB), isoprene oligomer (tc-E-DB), isoprene oligomer (tc- F-DB), used as an isoprene oligomer (tc-G-DB) in the following experiments.
In addition, a mutant enzyme N77A as an enzyme, a compound represented by the above formula (C) as a starting substrate, and IPP, R-IPP-A, R-IPP-B, R-IPP-C, R- as a monomer are used. The isoprene oligomer obtained by using IPP-D, R-IPP-E, R-IPP-F, and R-IPP-G, respectively, is isoprene oligomer (tc-control-AC), isoprene oligomer (tc- A-AC), isoprene oligomer (tc-B-AC), isoprene oligomer (tc-C-AC), isoprene oligomer (tc-D-AC), isoprene oligomer (tc-E-AC), isoprene oligomer (tc- F-AC), used as an isoprene oligomer (tc-G-AC) in the following experiments.
In addition, a mutant enzyme L91D as an enzyme, a compound represented by the above formula (C) as a starting substrate, and IPP, R-IPP-A, R-IPP-B, R-IPP-C, R- as a monomer are used. The isoprene oligomer obtained by using IPP-D, R-IPP-E, R-IPP-F, and R-IPP-G, respectively, is isoprene oligomer (tc-control-DC), isoprene oligomer (tc- A-DC), isoprene oligomer (tc-B-DC), isoprene oligomer (tc-C-DC), isoprene oligomer (tc-D-DC), isoprene oligomer (tc-E-DC), isoprene oligomer (tc- F-DC) and isoprene oligomer (tc-G-DC) were used in the following experiments.
In addition, isoprene oligomer (tc-E-AB), isoprene oligomer (tc-F-AB), isoprene oligomer (tc-E-DB), isoprene oligomer (tc-F-DB) are isoprene oligomer c, isoprene oligomer f The isoprene oligomer d and the isoprene oligomer g were blended in the rubber composition in the following experiment.

(Relative activity of each monomer)
Using each R-IPP prepared in IPP or Preparation Example 1 and farnesyl diphosphate (FPP) or allylic diphosphate derivatives prepared in Preparation Examples 2 and 3, the reaction is carried out under the following conditions to obtain FPP, The relative activity in the case of using each allylic diphosphate derivative and the case of using each R-IPP with respect to the case of using IPP was indexed with the activity of FPP and IPP as 100.
500 ng of each purified protein, 50 mM Tris-HCl buffer (pH 7.5), 40 mM magnesium chloride, 40 mM Triton X-100, 25 mM 2-mercaptoethanol, 12.5 μM FPP or allylic diphosphate prepared according to Preparation Examples 2 and 3 A reaction solution containing an acid derivative, 50 μM [1- ¹⁴ C] IPP or each R-IPP prepared in Preparation Example 1 was prepared, and reacted at 37 ° C. water bath for 1 hour. After the reaction, the activity under each condition is measured by quantifying the value of liquid scintillation and TLC, and the activity for each of FPP and IPP is 100, and each allylic diphosphate derivative and each R-IPP are used. The relative activities are shown in Table 11.
Also, the isoprene oligomer was purified in the same manner as described above (Example 7) (preparation of isoprene oligomer). And the detail (n, m, R in a formula (Z-2-1-1)) of the obtained isoprene oligomer was shown in Table 11. In the formula (Z-2-1-1), n and m were calculated based on the information on the starting substrate used and the isoprene chain length by TLC. Moreover, R in Formula (Z-2-1-1) identified the structure by NMR, TLC, and GC-MS.

From the results of Tables 10 and 11, even when R-IPP and allylic diphosphate derivatives were used, isoprene oligomers having the same molecular weight as when IPP and FPP were used were obtained. Further, it was also confirmed that the obtained isoprene oligomer was modified according to the R-IPP and allylic diphosphate derivative used.

(Example 8)
(Preparation of polyisoprene)
Next, an isoprene oligomer in which an isoprene unit represented by the above formula (Z-2-1-1) is bound in a trans-cis form using an allylic diphosphate derivative, is added to the main chain portion Polyisoprene was prepared using an isoprene oligomer (main chain terminal modified isoprene oligomer) in which the terminal portion was also modified.
10 μl of the latex component, 50 mM Tris-HCl buffer (pH 7.5), 25 mM magnesium chloride, 40 mM 2-mercaptoethanol, 40 mM potassium fluoride, 50 μM isoprene oligomer, 1 mM IPP or each R-IPP prepared in Preparation Example 1 The reaction solution was adjusted, and reacted at 30 ° C. water bath for 3 days. After the reaction, the molecular weight was measured by GPC. Then, based on the measured molecular weight and information on the used starting substrate, the number of isoprene units added to the starting substrate FPP or allylic diphosphate derivative was calculated. The results are shown in Table 12.

As a latex component, a serum prepared by ultracentrifugation of a latex obtained from Hevea brasiliensis was used.

The isoprene oligomer may be selected from isoprene oligomer (tc-control-AB), isoprene oligomer (tc-A-AB), isoprene oligomer (tc-B-AB), isoprene oligomer (tc) prepared by the above (preparation of isoprene oligomer). -C-AB), isoprene oligomer (tc-D-AB), isoprene oligomer (tc-E-AB), isoprene oligomer (tc-F-AB), isoprene oligomer (tc-G-AB), isoprene oligomer (tc -Control-DB), isoprene oligomer (tc-A-DB), isoprene oligomer (tc-B-DB), isoprene oligomer (tc-C-DB), isoprene oligomer (tc-D-DB), isoprene oligomer (tc) -E-DB), iso Ren oligomers (tc-F-DB), isoprene oligomers (tc-G-DB), isoprene oligomers (tc-control-AC), isoprene oligomers (tc-A-AC), isoprene oligomers (tc-B-AC), Isoprene oligomer (tc-C-AC), isoprene oligomer (tc-D-AC), isoprene oligomer (tc-E-AC), isoprene oligomer (tc-F-AC), isoprene oligomer (tc-G-AC), Isoprene oligomer (tc-control-DC), isoprene oligomer (tc-A-DC), isoprene oligomer (tc-B-DC), isoprene oligomer (tc-C-DC), isoprene oligomer (tc-D-DC), Isoprene oligomer (tc-E-DC), isopropyl Emissions oligomer (tc-F-DC), isoprene was used oligomer (tc-G-DC).
In addition, isoprene oligomer (tc-E-AB), isoprene oligomer (tc-F-AB), isoprene oligomer (tc-E-DB), isoprene oligomer (tc-F-DB), isoprene oligomer (tc-E-AC) And polyisoprene E obtained by using isoprene oligomer (tc-E-DC) as polyisoprene E, polyisoprene B, polyisoprene F, polyisoprene C, polyisoprene G, and polyisoprene H, the following experiments Were blended into the rubber composition.

From the results of Table 12, even when R-IPP and main chain terminal modified isoprene oligomers were used, polyisoprene having the same molecular weight as when IPP and unmodified isoprene oligomers were used was obtained. Further, as a result of analyzing the obtained polyisoprene by NMR, TLC and GC-MS, it is found that the polyisoprene is modified according to the R-IPP used and the main chain terminal modified isoprene oligomer as in the case of the isoprene oligomer. I also confirmed that.

The isoprene oligomer prepared in the above example, the terminal part of the polyisoprene (the above formula (Z-1), the above formula (Z-2), the above formula (ZZ-1), the above formula (ZZ-2), etc. (Equivalent to Y) was a hydroxyl group or an OPP group.

Hereinafter, various medicines used in Examples 9 to 41 and Comparative Examples 1 to 4 will be collectively described.
NR: TSR20
BR: BR01 manufactured by JSR Corporation
Carbon black: Diamond black (N220) manufactured by Mitsubishi Chemical Corporation
Isoprene oligomers a to n: isoprene oligomer polyisoprenes A to P obtained in the above example: polyisoprene zinc obtained in the above example: zinc oxide No. 1 stearic acid manufactured by Mitsui Metal Mining Co., Ltd. stearic acid: NOF Stearic acid anti-aging agent manufactured by Kabushiki Kaisha: Noclac 6C (N- (1,3-dimethylbutyl) -N'-phenyl-p-phenylenediamine) manufactured by Ouchi Emerging Chemical Industry Co., Ltd.
Wax: Sunnock wax sulfur manufactured by Ouchi Emerging Chemical Industry Co., Ltd. Powder sulfur vulcanization accelerator NS manufactured by Tsurumi Chemical Co., Ltd. NS: Noxella NS manufactured by Ouchi Emerging Chemical Industry Co., Ltd. (N-tert- Butyl-2-benzothiazylsulfenamide)
Silica: Nip seal AQ (wet silica) manufactured by Nippon Silica Kogyo Co., Ltd.
Silane coupling agent: Si266 (bis (3-triethoxysilylpropyl) disulfide) manufactured by Degussa
Vulcanization accelerator DPG: Noxceler D (N, N-diphenylguanidine) manufactured by Ouchi Shinko Chemical Co., Ltd.

According to the compounding prescription shown to Tables 13-16, materials other than sulfur and a vulcanization accelerator were knead | mixed using a 1.7 L Banbury mixer, and the kneaded material was obtained. Next, sulfur and a vulcanization accelerator were added to the obtained kneaded product, and the mixture was kneaded using an open roll to obtain an unvulcanized rubber composition. The obtained unvulcanized rubber composition was vulcanized at 150 ° C. for 30 minutes at a pressure of 80 kgf / cm ² using a steam vulcanization press to obtain a vulcanized rubber composition.

The following evaluation was performed about the obtained vulcanized rubber composition. The results are shown in Tables 13-16. The reference composition in Table 13 is Comparative Example 1; the reference composition in Table 14 is Comparative Example 2; the reference composition in Table 15 is Comparative Example 3; and the reference composition in Table 16 is Comparative Example 4.

(Viscoelastic test)
The tan δ was measured at 70 ° C. and a strain of 2% (initial elongation) at 70 ° C. using a viscometer manufactured by Iwamoto Seisakusho Co., Ltd., and the tan δ of the standard composition was expressed as an index of 100. The larger the index, the greater the heat generation. The heat resistance (low heat buildup) was considered to be improved when the index was 100 or less. That is, the smaller the index, the better the low heat buildup.

(Rambone wear test)
A wear test was carried out for 5 minutes under the conditions of a load of 3 kg, a slip ratio of 40% and a sand amount of 15 g / min using a Lambourn wear tester manufactured by Iwamoto Manufacturing Co., Ltd. The shape of the sample was 5 mm in thickness and 50 mm in diameter, and the grindstone used was a GC type abrasive of grain size # 80. The test results were indexed as 100 (standard) for the standard formulation. The larger the index, the better the abrasion resistance, and when the index exceeded 100, the abrasion resistance was considered to be improved.

(Tensile test)
A tensile test is carried out using a No. 3 dumbbell-shaped test piece consisting of the above-mentioned vulcanized rubber sheet according to JIS K6251 "Vulcanized rubber and thermoplastic rubber-Determination of tensile properties", and breaking strength (TB) (MPa) ) And elongation at break (EB) (%) were measured. If the elongation at break is less than 480%, rubber chipping tends to occur when it is used for a large tire, and improvement is necessary. In addition, since the breaking strength is also a cause of breakage of the tire when it is reduced, it is necessary to prevent the deterioration due to the change of material.

From Tables 13 and 15, the examples using the isoprene oligomer of the present invention were excellent in low heat buildup, wear resistance and elongation at break.

From Tables 14 and 16, the examples using the polyisoprene of the present invention were excellent in low heat buildup, wear resistance and breaking strength.

(Sequence table free text)
SEQ ID NO: 1: sense primer for preparation of mutant enzyme N77A SEQ ID NO: 2: antisense primer for preparation of mutant enzyme N77A SEQ ID NO: 3: sense primer for preparation of mutant enzyme L91D SEQ ID NO 4: antisense primer for preparation of mutant enzyme L91D SEQ ID NO: 5: Amino acid sequence of mutant enzyme N77A SEQ ID NO: 6: amino acid sequence of mutant enzyme L91D

Claims (8)

It is an isoprene oligomer represented by a following formula (Z-1) or a following formula (Z-2), Comprising: The atom or atom contained in v part in a following formula (Z-1) or a following formula (Z-2) An isoprene oligomer wherein at least one of the groups is replaced by another atom or group.

(In the formula (Z-1), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

(In the formula (Z-2), n represents an integer of 1 to 10. m represents an integer of 1 to 30. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxy carboxy group, an alkoxy carbonyl group or an OPP group Represents The isoprene oligomer according to claim 1, wherein at least one atom or atomic group contained in the iv moiety in the formula (Z-1) or the formula (Z-2) is substituted by another atom or atomic group. A polyisoprene obtained by being synthesized from the isoprene oligomer according to claim 1 and a compound represented by the following formula (Y) and / or isopentenyl diphosphate.

(In formula (Y), R represents a group other than a methyl group.) It is a polyisoprene represented by a following formula (ZZ-1) or a following formula (ZZ-2), and the atom or atom contained in v part in a following formula (ZZ-1) or a following formula (ZZ-2) Polyisoprene in which at least one of the groups is replaced by another atom or group.

(In the formula (ZZ-1), n represents an integer of 1 to 10. q represents an integer of 30 to 40. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents

(In the formula (ZZ-2), n represents an integer of 1 to 10. q represents an integer of 30 to 40. Y represents a hydroxyl group, a formyl group, a carboxy group, an alkoxycarboxy group, an alkoxycarbonyl group or an OPP group Represents The polyisoprene according to claim 4, wherein at least one atom or atomic group contained in the iv moiety in the formula (ZZ-1) or the formula (ZZ-2) is substituted by another atom or atomic group. The manufacturing method of the polyisoprene synthesize | combined from the isoprene oligomer of Claim 1 or 2, and the compound represented by said Formula (Y), and / or isopentenyl diphosphate. A rubber composition comprising the isoprene oligomer according to claim 1 and / or the polyisoprene according to any one of claims 3 to 5. A pneumatic tire produced using the rubber composition according to claim 7.