CN108484663B - Isoprene oligomer and method for producing isoprene oligomer - Google Patents

Isoprene oligomer and method for producing isoprene oligomer Download PDF

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CN108484663B
CN108484663B CN201810340783.6A CN201810340783A CN108484663B CN 108484663 B CN108484663 B CN 108484663B CN 201810340783 A CN201810340783 A CN 201810340783A CN 108484663 B CN108484663 B CN 108484663B
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isoprene oligomer
isoprene
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CN108484663A (en
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井之上雪乃
大谷典正
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Sumitomo Rubber Industries Ltd
Yamagata University NUC
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Sumitomo Rubber Industries Ltd
Yamagata University NUC
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B60—VEHICLES IN GENERAL
    • B60C—VEHICLE TYRES; TYRE INFLATION; TYRE CHANGING; CONNECTING VALVES TO INFLATABLE ELASTIC BODIES IN GENERAL; DEVICES OR ARRANGEMENTS RELATED TO TYRES
    • B60C1/00—Tyres characterised by the chemical composition or the physical arrangement or mixture of the composition
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/113—Esters of phosphoric acids with unsaturated acyclic alcohols
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F36/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds
    • C08F36/02—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds the radical having only two carbon-to-carbon double bonds
    • C08F36/04—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, at least one having two or more carbon-to-carbon double bonds the radical having only two carbon-to-carbon double bonds conjugated
    • C08F36/08—Isoprene
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G61/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G61/02—Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes
    • C08G61/04—Macromolecular compounds containing only carbon atoms in the main chain of the macromolecule, e.g. polyxylylenes only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L15/00—Compositions of rubber derivatives
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L9/00—Compositions of homopolymers or copolymers of conjugated diene hydrocarbons
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G2261/00—Macromolecular compounds obtained by reactions forming a carbon-to-carbon link in the main chain of the macromolecule
    • C08G2261/30—Monomer units or repeat units incorporating structural elements in the main chain
    • C08G2261/33—Monomer units or repeat units incorporating structural elements in the main chain incorporating non-aromatic structural elements in the main chain
    • C08G2261/332—Monomer units or repeat units incorporating structural elements in the main chain incorporating non-aromatic structural elements in the main chain containing only carbon atoms
    • C08G2261/3327—Monomer units or repeat units incorporating structural elements in the main chain incorporating non-aromatic structural elements in the main chain containing only carbon atoms alkene-based

Abstract

The invention aims to provide a molecular skeleton modified isoprene oligomer and polyisoprene. It is also an object of the present invention to provide a rubber composition comprising the isoprene oligomer and/or the polyisoprene, and a pneumatic tire comprising a tire member (e.g., tread or sidewall) formed from the rubber composition. The present invention relates to an isoprene oligomer synthesized from allyl 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 the formula (Y), R represents a group other than methyl.

Description

Isoprene oligomer and method for producing isoprene oligomer
The present application is a divisional application based on the following chinese patent applications:
application date of the original case: 09 month and 02 days 2013
Original application No.: 201380045290.5(PCT/JP2013/073509)
The original application name: isoprene oligomer, polyisoprene, method for producing isoprene oligomer and polyisoprene, rubber composition, and pneumatic tire
Technical Field
The present invention relates to isoprene oligomers, polyisoprenes, methods for making isoprene oligomers and polyisoprenes, compositions containing isoprene oligomers and/or polyisoprene rubbers, and pneumatic tires formed from the rubber compositions.
Background
For a long time, efforts have been made to provide rubber products having new properties in addition to the original properties of rubber, and fillers or other components of different shapes made of different materials are introduced into rubber compositions to obtain desired properties depending on the use. For example, in automobile tires, attempts have been made to improve properties such as abrasion resistance, low heat build-up, and wet grip performance by introducing a filler such as silica or carbon black into an organic rubber phase.
Conventionally, a method of mixing a filler or the like in a rubber phase such as a rubber composition or a modified rubber (modified diene polymer) has been used to increase the affinity between these components and further improve the low heat build-up, wet grip performance and the like. These modified rubbers introduce a functional group having affinity with a filler into a rubber molecule by reacting the rubber molecule in a rubber phase with, for example, a compound containing a nitrogen-containing group, a chlorosulfonyl group (for example, refer to patent documents 1 and 2).
Meanwhile, isoprene oligomers and polyisoprene are known to have a diphosphate group or the like having high reactivity at the chain end thereof. Therefore, the conventional method for modifying isoprene oligomer or polyisoprene involves the reaction of the diphosphate group at the highly reactive terminus described above with a functional group-containing compound. As described above, isoprene oligomers and polyisoprene have been conventionally modified mainly at the terminal ends of the molecule. On the other hand, in the conventional method, the skeleton of isoprene oligomer and polyisoprene cannot be sufficiently modified by the influence of the diphosphate group or the like at the highly reactive terminal. Furthermore, conventionally, modification of natural rubber can only be accomplished by chemical treatment of latex collected from Hevea brasiliensis (Hevea brasiliensis). Therefore, it cannot be confirmed by analysis whether the molecular chain is actually modified and what type of functional group is substituted.
List of cited documents
Patent document
Patent document 1: JP 2000 + 001573A
Patent document 2: JP 2000 + 001575A
Disclosure of Invention
Technical problem
The object of the present invention is to solve the above problems and to provide a molecular skeleton-modified isoprene oligomer and polyisoprene. It is also an object of the present invention to provide a rubber composition comprising the isoprene oligomer and/or the polyisoprene, and a pneumatic tire comprising a tire member (e.g., tread or sidewall) formed from the rubber composition.
Means for solving the problems
The present invention relates to an isoprene oligomer synthesized from allyl diphosphate represented by the formula (X) and a compound represented by the formula (Y):
Figure BDA0001630113170000021
wherein n represents an integer of 1 to 10;
Figure BDA0001630113170000022
wherein R represents a group other than a methyl group.
Preferably, the isoprene oligomer is synthesized from the allyl diphosphate represented by the formula (X), the compound represented by the formula (Y), and isopentenyl diphosphate.
Preferably, the allyl diphosphate represented by the formula (X) is an allyl diphosphate represented by the formula (X-1):
Figure BDA0001630113170000031
wherein n represents an integer of 1 to 10, and at least one atom or group of atoms contained in the moiety II in formula (X-1) is substituted with another (i.e., different) atom or group of atoms, and none of the atoms or groups of atoms contained in the moiety III in formula (X-1) is substituted with another atom or group of atoms.
The synthesis is preferably carried out by using an enzyme having prenyl transferase activity.
The present invention also relates to an isoprene oligomer represented by the formula (Z-1) or the formula (Z-2), wherein at least one atom or atomic group contained in the moiety v in the formula (Z-1) or the formula (Z-2) is substituted with another atom or atomic group.
Figure BDA0001630113170000032
Wherein n in the formula (Z-1) represents an integer of 1 to 10; m represents an integer of 1 to 30; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP;
Figure BDA0001630113170000033
wherein n in the formula (Z-2) represents an integer of 1 to 10; m represents an integer of 1 to 30; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP.
It is preferred that at least one atom or group of atoms contained in the moiety iv in formula (Z-1) or formula (Z-2) is substituted with another atom or group of atoms.
The present invention also relates to a method for producing an isoprene oligomer, which comprises synthesizing an isoprene oligomer from allyl diphosphate represented by the formula (X) and a compound represented by the formula (Y):
Figure BDA0001630113170000041
wherein n represents an integer of 1 to 10;
Figure BDA0001630113170000042
wherein R represents a group other than a methyl group.
In the method for producing an isoprene oligomer, it is preferable that the isoprene oligomer is synthesized from the allyl diphosphate represented by the formula (X), the compound represented by the formula (Y), and isopentenyl diphosphate.
Preferably, the allyl diphosphate represented by the formula (X) is an allyl diphosphate represented by the formula (X-1):
Figure BDA0001630113170000043
wherein n represents an integer of 1 to 10, and at least one atom or atom group contained in the moiety II in the formula (X-1) is substituted with another atom or atom group, and none of the atoms or atom groups contained in the moiety III in the formula (X-1) is substituted with another atom or atom group.
The synthesis is preferably carried out by using an enzyme having prenyl transferase activity.
The invention also relates to polyisoprene which is synthesized by allyl diphosphate represented by a formula (X) and a compound represented by a formula (Y):
Figure BDA0001630113170000044
wherein n represents an integer of 1 to 10;
Figure BDA0001630113170000051
wherein R represents a group other than a methyl group.
Preferably, the polyisoprene is synthesized from the allyl diphosphate represented by the formula (X), the compound represented by the formula (Y), and isopentenyl diphosphate.
Preferably, the allyl diphosphate represented by the formula (X) is an allyl diphosphate represented by the formula (X-1):
Figure BDA0001630113170000052
wherein n represents an integer of 1 to 10, and at least one atom or atom group contained in the moiety II in the formula (X-1) is substituted with another atom or atom group, and none of the atoms or atom groups contained in the moiety III in the formula (X-1) is substituted with another atom or atom group.
The invention also relates to polyisoprene which is synthesized by the isoprene oligomer, at least one compound represented by the formula (Y) and isopentenyl diphosphate.
The invention also relates to a polyisoprene of formula (ZZ-1) or (ZZ-2), wherein at least one atom or group of atoms contained in the moiety v of formula (ZZ-1) or (ZZ-2) is replaced by another atom or group of atoms,
Figure BDA0001630113170000053
wherein n in the formula (ZZ-1) represents an integer of 1 to 10; q represents an integer of 30 to 40000; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP;
Figure BDA0001630113170000061
wherein n in the formula (ZZ-2) represents an integer of 1 to 10; q represents an integer of 30 to 40000; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP.
Preferably, at least one atom or group of atoms contained in moiety iv in formula (ZZ-1) or formula (ZZ-2) is replaced by another atom or group of atoms.
The present invention also relates to a method for producing polyisoprene, which comprises synthesizing the polyisoprene from the isoprene oligomer, at least one compound represented by the formula (Y), and isopentenyl diphosphate.
The present invention also relates to a rubber composition comprising at least one of the isoprene oligomer and the polyisoprene.
The present invention relates to a pneumatic tire formed from the rubber composition.
Advantageous effects of the invention
The isoprene oligomer of the present invention is an isoprene oligomer synthesized from allyl diphosphate represented by formula (X) and a compound represented by formula (Y), or it is an isoprene oligomer represented by formula (Z-1) or formula (Z-2), wherein at least one atom or atomic group contained in the moiety v in formula (Z-1) or formula (Z-2) is substituted with another atom or atomic group. Further, the polyisoprene of the present invention is polyisoprene synthesized from allyl diphosphate represented by the formula (X) and a compound represented by the formula (Y); or a polyisoprene synthesized from the above-mentioned isoprene oligomer, a compound represented by the formula (Y) and/or isopentenyl diphosphate; or a polyisoprene represented by formula (ZZ-1) or formula (ZZ-2), wherein at least one atom or group of atoms contained in the v moiety in formula (ZZ-1) or formula (ZZ-2) is substituted with another atom or group of atoms. Therefore, the isoprene oligomer of the present invention and the polyisoprene of the present invention (rubber molecule) each have a modified skeleton, and thus have excellent affinity with a filler such as silica. Therefore, if the isoprene oligomer of the present invention and/or the polyisoprene of the present invention is used for a rubber composition, the rubber molecules in the obtained rubber composition can be highly mixed with a filler. For example, the rubber composition thus provided has excellent low heat build-up and abrasion resistance. Further, for example, if the above rubber composition is used in a tire component (e.g., tread or sidewall), a pneumatic tire having excellent low heat build-up and wear resistance can be provided.
Further, the method for producing an isoprene oligomer of the present invention and the method for producing polyisoprene of the present invention can provide an isoprene oligomer and polyisoprene, respectively, which are sufficiently modified in the skeleton without affecting the high-reactive terminal diphosphate group.
Further, since the isoprene oligomer and polyisoprene provided by the present invention are obtained using the compound represented by formula (Y) to which a functional group or other group having a known structure is added, the functional group (modifying group) in the isoprene oligomer or polyisoprene can be specifically identified.
Detailed Description
In the artificial synthesis (biosynthesis) of a rubber molecule (polyisoprene), an enzyme (e.g., prenyltransferase) can be used to act on a mixture of a starting substrate (e.g., farnesyl diphosphate (FPP)) and a monomer (e.g., isopentenyl diphosphate) to produce an isoprene oligomer having 1 to 30 isoprene units addition-polymerized on the starting substrate. Next, these isoprene oligomers are further mixed with a latex component containing an enzyme capable of addition-polymerizing prenyl diphosphates. It is known that the result is the formation of polyisoprene having a number of polyisoprene units attached to the oligomer.
As mentioned above, the step of continuously linking monomers on the starting substrate to form the rubber molecule must use a natural enzyme catalyzed addition polymerization.
This means that the starting substrates and monomers for the synthesis of the rubber molecule (polyisoprene) need to be substances which can be reacted catalytically with enzymes. Therefore, the structures of starting substrates and monomers that can be used as materials for rubber molecules (polyisoprene) are limited. In particular, monomers are limited to the naturally occurring prenyl diphosphates because of limitations imposed by prenyltransferases and enzymes capable of addition polymerization of prenyl diphosphates.
As a result, the structural design flexibility of the artificially synthesized (biosynthetic) rubber molecule (polyisoprene) is limited, and it is therefore difficult to provide sufficient flexibility in molecular design to add properties that natural rubber does not have.
For these reasons, in order to prepare a rubber molecule (polyisoprene) into which a functional group or the like is introduced, for example, conventionally, as described above, one causes modification by, for example, reacting a highly reactive terminal diphosphate group or the like with a functional group-containing compound. In particular, the modification of natural rubber can only be accomplished by chemical treatment of latex collected from Hevea brasiliensis (Hevea brasiliensis). Therefore, it cannot be confirmed by analysis whether the molecular chain is actually modified and what type of functional group is substituted.
In contrast, the present invention is based on the following findings: by producing an isoprene oligomer or polyisoprene using isopentenyl diphosphate having a partially modified structure as a monomer, an isoprene oligomer or polyisoprene having a functionalized skeleton can be produced. Further, since the isoprene oligomer and polyisoprene provided by the present invention are obtained using isopentenyl diphosphate to which a functional group or other group having a known structure is added, the functional group (modifying group) in the isoprene oligomer or polyisoprene can be specifically identified.
In particular, the invention is based on the finding that: if the structure other than the methyl group at the 3-position in the structure of the naturally occurring monomer isopentenyl diphosphate is maintained, even when the methyl group at the 3-position is substituted by a desired group, the monomer can be used to produce an isoprene oligomer or polyisoprene by prenyltransferase which is a naturally occurring oligomer-producing enzyme or a naturally occurring polymer-producing enzyme capable of addition-polymerizing isopentenyl diphosphate. The reason for this is not fully elucidated, but it is presumed that prenyl transferases and enzymes capable of addition polymerization of prenyl diphosphate are adsorbed to structures other than the methyl group at the 3-position of prenyl diphosphate monomer, and these enzymes are relatively insensitive to the structure of the methyl group at the 3-position.
Based on these findings, it is possible to provide isoprene oligomers and polyisoprenes having a skeleton with desired properties, and thus it is possible to provide isoprene oligomers and polyisoprenes having various additional functions.
More specifically, in naturally occurring biosynthesis, for example, isopentenyl diphosphate (IPP) may be continuously polymerized on dimethylallyl Diphosphate (DMAPP), thereby producing geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (GFPP), as shown below.
Figure BDA0001630113170000091
Scheme 1
In contrast, the present invention uses R-IPP obtained by substituting the methyl group at the 3-position of IPP with a desired group (-R) instead of isopentenyl diphosphate (IPP). In this case, R-IPP may be continuously polymerized on dimethylallyl diphosphate, thereby yielding backbone-modified geranyl diphosphate (R-GPP), backbone-modified farnesyl diphosphate (R-FPP), backbone-modified geranylgeranyl diphosphate (R-GGPP), backbone-modified geranylfarnesyl diphosphate (R-GFPP), as shown below.
Figure BDA0001630113170000101
Scheme 2
Further, if R-IPP is used simultaneously with IPP, in other words, R-IPP and IPP are used in combination, and R-IPP and IPP are polymerized continuously on dimethylallyl diphosphate, backbone-modified geranyl diphosphate (R-GPP), backbone-modified farnesyl diphosphate (R-FPP), backbone-modified geranylgeranyl diphosphate (R-GGPP), backbone-modified geranylfarnesyl diphosphate (R-GFPP) as shown below can be produced. Therefore, an isoprene oligomer having desired properties can be produced by using R-IPP and IPP in combination in the adjusted ratio.
Figure BDA0001630113170000111
Scheme 3
Here, the modification of the skeleton of the molecule (rubber molecule) means that a structure derived from IPP (specifically, a portion corresponding to the methyl group at the 3-position of IPP) in the skeleton of the molecule (rubber molecule) is substituted with a desired functional group, or that a structure derived from IPP (specifically, a portion corresponding to the methyl group at the 3-position of IPP) in the skeleton of the molecule (rubber molecule) is substituted with another structure (a structure other than a methyl group).
(isoprene oligomer)
An oligomer of the present invention is synthesized (biosynthesized) from allyl diphosphate represented by the formula (X) and a compound (R-IPP) represented by the formula (Y):
Figure BDA0001630113170000121
wherein n represents an integer of 1 to 10;
Figure BDA0001630113170000122
wherein R represents a group other than a methyl group.
Here, the term "OPP (OPP group)" means a diphosphate group (a group represented by the following formula (a-1)), and it has 3 hydroxyl groups bonded to phosphorus atoms. When it is in an aqueous solution, a part or all of the hydroxyl groups are dissociated (for example, OPP becomes a group represented by the following formula (A-2)). The term "OPP" as used herein includes groups wherein some or all of the hydroxyl groups are cleaved.
Figure BDA0001630113170000123
The isoprene oligomer of the present invention has a structure similar to that of natural rubber and is highly compatible with rubber molecules. In addition, since the isoprene oligomer of the present invention has a modified skeleton, it can strongly interact with a filler (e.g., silica). Therefore, since the isoprene oligomer of the present invention is highly compatible with rubber while being strongly interactive with a filler (e.g., silica), if the isoprene oligomer is used for a rubber composition, a rubber composition in which rubber molecules are mixed with a filler to a higher degree than in conventional compositions can be obtained. For example, the rubber composition can thereby enhance low heat build-up, wet grip performance, abrasion resistance, elongation at break, and tensile strength at break.
The skeleton of the isoprene oligomer of the present invention contains a polar group or the like. Thus, the dispersing property of the filler (e.g., silica) is higher than that in the case where a polar group or the like is present only at the chain end. Therefore, for example, the improvement effects of low heat build-up, wet grip performance, abrasion resistance, elongation at break and tensile strength at break are increased.
The isoprene oligomers of the present invention also have excellent antimicrobial activity. This is presumably because the isoprene oligomer of the present invention has a structure different from that of a naturally occurring normal isoprene oligomer, and therefore has effects such as inhibition of bacterial enzymes and coenzymes, inhibition of nucleic acid synthesis, inhibition of cell membrane synthesis, inhibition of synthesis of cytoplasmic membranes, damage to cell membranes, and damage to cytoplasmic membranes.
First, allyl diphosphate represented by the formula (X) will be described:
Figure BDA0001630113170000131
wherein n represents an integer of 1 to 10.
In the formula (X), n represents an integer of 1 to 10 (preferably 1 to 4, more preferably 1 to 3).
Examples of the allyl diphosphate represented by the formula (X) include: dimethylallyl Diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), geranylfarnesyl diphosphate (GFPP). Among them, DMAPP, GPP and FPP are preferable because they can serve as substrates for various types of prenyl transferases.
In the allyl diphosphate represented by the formula (X), a diphosphate group is bonded to an isoprene unit. The allyl diphosphate represented by the formula (X) may be an allyl diphosphate derivative in which a part of an isoprene unit is changed (modified).
The present inventors have proposed a patent application for a method for producing an isoprene oligomer or polyisoprene using an allyl diphosphate derivative as a starting substrate (JP2012-036360 a). This patent application is briefly described below.
As described above, the starting substrate and monomer for the synthesis of the rubber molecule (polyisoprene) need to be substances that can be subjected to a catalytic reaction with an enzyme. This limits the structures of the starting substrates and monomers that can be used as materials for rubber molecules (polyisoprene). In particular, because of the limitations imposed by the enzymes making the oligomers, the starting substrates are limited to the naturally occurring dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, and the like.
In contrast, the present inventors have found through extensive studies that an isoprene oligomer or polyisoprene having a functionalized chain end can be produced by producing an isoprene oligomer or polyisoprene using, for example, farnesyl diphosphate or the like whose structure is partially modified as a starting substrate.
In particular, the present inventors have found that if the structure of the moiety I in the following formula (I) in a naturally occurring starting substrate such as farnesyl diphosphate is maintained, an isoprene oligomer can be produced using prenyltransferase which is a naturally occurring oligomer-producing enzyme even if a desired structure is introduced into a portion other than the moiety I. The reason for this is not fully elucidated, but it is presumed that the prenyltransferase is adsorbed on the structure of the I moiety in formula (I) of the starting substrate, while these enzymes are relatively insensitive to the structure of other moieties.
Figure BDA0001630113170000141
Based on these findings, it is possible to provide an isoprene oligomer and polyisoprene having a chain end with a desired property, and therefore, it is possible to provide an isoprene oligomer and polyisoprene having various functions added thereto without impairing the original properties of the isoprene oligomer or polyisoprene.
Therefore, in the present invention, by polymerizing the compound of formula (Y) (R-IPP) onto the starting substrate, which is the allyl diphosphate of formula (X) modified while maintaining the partial structure I of formula (I), using prenyltransferase of a naturally occurring oligomer-producing enzyme, an isoprene oligomer in which not only the backbone but also the chain end (the moiety derived from allyl diphosphate) is modified can be obtained.
In particular, it is preferable that the allyl diphosphate represented by the above formula (X) is an allyl diphosphate represented by the formula (X-1):
Figure BDA0001630113170000142
wherein n represents an integer of 1 to 10 (n in formula (X-1) is defined in the same manner as in formula (X)), and at least one atom or atomic group contained in the moiety II in formula (X-1) is substituted with another atom or atomic group, and none of the atoms or atomic groups contained in the moiety III in formula (X-1) is substituted with another atom or atomic group.
As a result, an isoprene oligomer having not only a modified skeleton but also modified chain ends was produced. Since the isoprene oligomer has not only a modified skeleton but also modified chain ends, it can better interact with a filler (e.g., silica), and if the isoprene oligomer is used for a rubber composition, the rubber molecules in the obtained rubber composition can be mixed with the filler to a higher degree than in a conventional composition. For example, the rubber composition can thereby provide further enhanced low heat build-up, wet grip performance, abrasion resistance, elongation at break, and tensile strength at break.
Here, the modification of the terminal of the molecule (rubber molecule) means that a predetermined portion derived from the allyl diphosphate structure represented by the formula (X) at the terminal of the molecule (rubber molecule) is substituted with a desired functional group, or means that a predetermined portion derived from the allyl diphosphate structure represented by the formula (X) at the terminal of the molecule (rubber molecule) is substituted with another structure.
Examples of the atom or atom group (i.e., the atom or atom group before substitution) contained in the moiety II or the moiety III in the formula (X-1) include a hydrogen atom, a methyl group, a methylene group, a carbon atom and a methine group.
Examples of the other atoms include a nitrogen atom, an oxygen atom, a sulfur atom, a silicon atom and a carbon atom. Among them, nitrogen atoms are preferable because they have strong intermolecular forces and thus can have strong interactions with enzymes or cell membranes, and also have strong interactions with fillers (e.g., silica).
The other atomic group may be a nitrogen-containing group, an oxygen-containing group, a sulfur-containing group, a silicon-containing group, a carbon-containing group, or the like. Examples thereof include: acetoxy, alkoxy (preferably C1-C3 alkoxy, more preferably methoxy), hydroxy, aryl (preferably phenyl), alkyl (preferably C1-C5 alkyl, more preferably ethyl and tert-butyl), acetyl, N-alkyl-acetamido (wherein the alkyl group preferably has 1 to 5 carbon atoms) and azido.
From the viewpoint of antimicrobial ability, N-alkyl-acetylamino groups (more preferably N-methyl-acetylamino group and N-butyl-acetylamino group) and azido groups are preferable because nitrogen atoms have strong intermolecular forces and thus can strongly interact with enzymes or cell membranes. Alkoxy and hydroxyl groups are also preferred because of their strong interaction with the filler (e.g., silica).
Examples of the allyl diphosphate (allyl diphosphate derivative) modified with a part of isoprene units include compounds represented by the following formulae (a) to (S). Among them, the compounds of the formulae (B), (C), (D), (E), (F), (K), (L) and (R) are preferable, and the compounds of the formulae (B) and (C) are more preferable because they have stronger interaction with the filler (e.g., silica) and can more effectively improve low heat buildup, wet grip performance, abrasion resistance, elongation at break and tensile strength at break. The structures of the formulae (G) to (Q) are also preferable, and the structures of the formulae (K), (L) and (Q) are more preferable, based on excellent antimicrobial ability.
Figure BDA0001630113170000161
The skilled person can prepare the allyl diphosphate derivatives represented by formulae (a) to (S) according to the method disclosed in JP2012-036360a, for example, from dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, geraniol, farnesol, geranylgeraniol or the like.
Next, a compound (R-IPP) represented by the formula (Y) will be described:
Figure BDA0001630113170000162
wherein R represents a group other than a methyl group.
In the formula (Y), R may be any group other than methyl. Examples thereof include nitrogen-containing groups, oxygen-containing groups, sulfur-containing groups, silicon-containing groups and carbon-containing groups (other than methyl groups). Preferred examples include acetoxy, alkoxy, hydroxy, aryl, alkyl (other than methyl), acetyl, N-acetyl-acetamido, azido, amino, and mercapto. Among them, nitrogen-containing groups, oxygen-containing groups, sulfur-containing groups, silicon-containing groups and carbon-containing groups (except for methyl groups) are preferable, alkyl groups (particularly, C2-C6 alkyl groups), aryl groups (particularly, phenyl groups), mercapto groups, hydroxyl groups and amino groups are more preferable, and mercapto groups and hydroxyl groups are further preferable because they can more effectively improve low heat buildup, wet grip performance, elongation at break and tensile strength at break.
The skilled person can prepare the compound represented by the formula (Y) according to, for example, the methods described in the examples.
The number of addition of isoprene units to the allyl diphosphate starting substrate of formula (X) in the isoprene oligomer of the present invention is preferably 1 to 30, more preferably 1 to 20, further preferably 1 to 10.
For example, a specific example of the isoprene oligomer of the present invention is an isoprene oligomer represented by the formula (Z-1) or the formula (Z-2), wherein at least one atom or atomic group contained in the moiety v in the formula (Z-1) or the formula (Z-2) is substituted with another atom or atomic group.
Figure BDA0001630113170000171
Wherein n in the formula (Z-1) represents an integer of 1 to 10; m represents an integer of 1 to 30; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP;
Figure BDA0001630113170000172
wherein n in the formula (Z-2) represents an integer of 1 to 10; m represents an integer of 1 to 30; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP.
The atom or atomic group contained in the moiety v in formula (Z-1) or formula (Z-2) and the other atom or atomic group may be an atom or atomic group as described in formula (X-1).
The moiety iv in the formulae (Z-1) and (Z-2) is derived from the structure of the allyldiphosphonic acid starting substrate represented by the formula (X). Therefore, n in the formulae (Z-1) and (Z-2) is defined in the same manner as n in the formula (X).
The moiety v in the formulae (Z-1) and (Z-2) is composed of an isoprene unit added to the allyl diphosphate starting substrate represented by the formula (X). Thus, m in the formulae (Z-1) and (Z-2) corresponds to the number of isoprene units added to the allyldiphosphonic acid starting substrate of the formula (X) as described above.
Y in the formulae (Z-1) and (Z-2) is preferably OPP, hydroxyl group or carboxyl group because of excellent antimicrobial properties and strong interaction with a filler (e.g., silica).
As described above, the present invention is based on the following findings: if the structure other than the methyl group at the 3-position in the isopentenyl diphosphate structure is maintained, an isoprene oligomer or polyisoprene can be produced by a naturally occurring enzyme using the same even if the methyl group at the 3-position is substituted with a desired group. Accordingly, the isoprene oligomer represented by the formula (Z-1) or the formula (Z-2) is preferably an isoprene oligomer represented by the formula (Z-1-1) or the formula (Z-2-1):
Figure BDA0001630113170000181
wherein n, m, and Y in formula (Z-1-1) are defined in the same manner as n, m, and Y in formula (Z-1), and R is defined in the same manner as R in formula (Y);
Figure BDA0001630113170000182
wherein n, m, and Y in formula (Z-2-1) are defined in the same manner as n, m, and Y in formula (Z-2), and R is defined in the same manner as R in formula (Y);
as described above, a substance in which both the skeleton and the chain ends are modified is also preferable. Therefore, it is preferable that at least one atom or atom group contained in the moiety iv in the formula (Z-1) or the formula (Z-2) (preferably the formula (Z-1-1) or the formula (Z-2-1)) is substituted with another atom or atom group.
The atom or atomic group contained in the moiety iv in the formula (Z-1), the formula (Z-2), the formula (Z-1-1) or the formula (Z-2-1) and the other atom or atomic group may be as described in the formula (X-1).
Further, as described above, it is preferable that the allyl diphosphate represented by the formula (X) is an allyl diphosphate represented by the formula (X-1), and that at least one atom or atomic group contained in the moiety II in the formula (X-1) is substituted with another atom or atomic group, and that none of the atoms or atomic groups contained in the moiety III in the formula (X-1) is substituted with another atom or atomic group. Accordingly, with respect to the moiety iv in the formula (Z-1), the formula (Z-2), the formula (Z-1-1) and the formula (Z-2-1) represented by the following formula (Z), it is preferable that at least one atom or atomic group contained in the moiety iv-II in the formula (Z) is substituted with another atom or atomic group, and none of the atoms or atomic groups contained in the moiety iv-III in the formula (Z) is substituted with another atom or atomic group.
Figure BDA0001630113170000191
The atom or atomic group contained in the moiety iv-II or iv-III in the formula (Z) and the other atom or atomic group may be an atom or atomic group as described in the formula (X-1).
Specific examples of the iv moiety in which the chain end is also modified may include structures represented by the following formulae (a) to(s). Among them, the structures of the formulae (b), (c), (d), (e), (f), (k), (l) and (r) are preferable, and the structures of the formulae (b) and (c) are more preferable because they have stronger interaction with the filler (e.g., silica) and can more effectively improve low heat buildup, wet grip performance, abrasion resistance, elongation at break and tensile strength at break. Structures of formulae (g) to (q) are also preferred, more preferably structures of formulae (k), (l) and (q), due to excellent antimicrobial ability.
Figure BDA0001630113170000201
(method for producing isoprene oligomer)
The isoprene oligomer of the present invention is synthesized (biosynthesized) from allyl diphosphate represented by the formula (X) and a compound represented by the formula (Y).
As an example of the method for synthesizing (biosynthesizing) the isoprene oligomer of the present invention from allyl diphosphate represented by the formula (X) and a compound represented by the formula (Y), there is used an enzyme having prenyl transferase activity. Specifically, allyl diphosphate represented by the formula (X) is reacted with a compound represented by (Y) which is a monomer polymerizable on the acid form of allyl diphosphate in the presence of an enzyme having prenyl transferase activity. The allyl diphosphate represented by the formula (X) and the compound represented by the formula (Y) may be each a single compound or a combination of a plurality of compounds. For example, a combination of compounds of formula (Y) in which R varies in formula (Y) may be used to provide isoprene oligomers with desired properties.
In the present invention, the monomer polymerized on the allyl diphosphate represented by the formula (X) may be only the compound represented by the formula (Y) (R-IPP). In this case, the isoprene oligomer shown in scheme 2 can be produced. Alternatively, the monomer polymerized on the allyl diphosphate represented by the formula (X) may be a combination of the compound represented by the formula (Y) (R-IPP) and isopentenyl diphosphate (IPP). In this case, the isoprene oligomer shown in scheme 3 can be produced.
Here, the term "having prenyl transferase activity" means an enzyme having an activity of catalyzing a condensation reaction of an allyl substrate (allyl diphosphate) and isopentenyl diphosphate to synthesize new allyl diphosphate having an added isoprene unit, and thus, can catalyze a reaction of continuously linking isopentenyl diphosphate to an allyl substrate (allyl diphosphate).
As described above, when the compound of formula (Y) (R-IPP) is obtained by substituting the methyl group at the 3-position in the prenyl diphosphate structure with a desired group while maintaining the structure of the prenyl diphosphate other than the methyl group at the 3-position, it is possible to produce an isoprene oligomer by using the compound and using a naturally occurring enzyme such as an enzyme having prenyl transferase activity.
As enzymes having prenyl transferase activity, various types of enzymes have been found.
Enzymes capable of elongating the Z-isoprene chain (each newly added isoprene unit has a cis configuration) include: z-nonaprenyl diphosphate synthase (Ishii, K.et., (1986) biochem., J.233, 773.), undecaprenyl diphosphate synthase (Takahashi, I. and Ogura, K., (1982) J.biochem., 92, 1527.; Keenman, M.V. and Allen, C.M., (1974) Arch.biochem.biophysis, 161, 375.), Z-farnesyl diphosphate synthase (Identification OF a short-chain (C-15) gene Z-isoprenyl diphosphate synthesis and homology (C-50) gene in isoprenyl diphosphate synthase synthesis and dehydrogenase (C-50) gene OF a short-chain (C-15) gene coding subunit dehydrogenase in Mycobacterium tuberculosis (Bacillus), and (Identification OF long-chain dehydrogenase OF the same-chain diphosphate synthase, III) gene OF Escherichia coli dehydrogenase (C-15) and long-chain dehydrogenase (III-2) gene coding subunit dehydrogenase (C-15) genes), and (Identification OF long-chain dehydrogenase OF Escherichia coli dehydrogenase gene OF Escherichia coli dehydrogenase (I.2225, III), endo, Shota. et al, Biochimica et Biophysica Acta (BBA), 1625(3), (2003) p.291-295.).
In addition, enzymes capable of extending the E-prenyl chain (each newly added isoprene unit having a trans configuration) include: farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase, heptaprenyl diphosphate synthase, octaprenyl diphosphate synthase, and decaprenyl diphosphate synthase.
The maximum number of isoprene units that can be formed and the direction of isoprene chain elongation (trans or cis configuration) depends on the particular enzyme. Thus, the enzyme may be selected according to the target number of isoprene units and the isoprene chain elongation direction. In the present invention, the isoprene chain of the isoprene oligomer may be extended in any direction (trans configuration or cis configuration). In other words, for example, the isoprene oligomer of the present invention may be an isoprene oligomer in which all isoprene units are linked in a trans configuration (for example, an isoprene oligomer of formula (Z-1)), an isoprene oligomer in which isoprene units are linked in a trans-cis configuration (for example, an isoprene oligomer of formula (Z-2)), or an isoprene oligomer in which isoprene units are linked in a trans-cis-trans configuration.
Every organism present on earth possesses an enzyme with prenyl transferase activity. Examples of organisms having an enzyme having prenyl transferase activity include Micrococcus luteus B-P26(Micrococcus luteus B-P26), Escherichia coli (Escherichia coli), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Arabidopsis thaliana (Arabidopsis thaliana), Hevea brasiliensis (Hevea brasiliensis), Acanthopanacis cortex (Periplocaceae), Bacillus Stearothermophilus (Bachiatus stearthermophilus), Sulfolobus acidocaldarius (Sulfolobus acidicaldarius (ATCC49426)), Homo sapiens (Homo sapiens), Sonchus arvensis (Sonchus soralens L.), Taraxacum officinale (Taraxacum officinale) and Helianthus annuus.
The original substrate (starting substrate) of the enzyme having prenyl transferase activity is allyl diphosphate. Then, since the allyl diphosphate derivative originally acts as an inhibitor of the enzyme, in many cases, if the allyl diphosphate derivative is used as a starting substrate, the enzyme exhibits a low enzymatic activity on the allyl diphosphate derivative (in particular, compounds represented by formulae (G) to (Q)). For these reasons, in the case of using an allyl diphosphate derivative as a starting substrate, a variant enzyme whose enzymatic activity is enhanced can be used for the allyl diphosphate derivative. In the case of using a variant enzyme, an organism (transformant) transformed by a genetic engineering technique may be prepared to express the variant enzyme. In particular, the skilled person can easily prepare variant enzymes that increase the enzymatic activity of allyl diphosphate derivatives according to the method disclosed in JP2012-036360 a.
The isoprene oligomer of the present invention can be produced by reacting allyl diphosphate represented by the formula (X) with a compound represented by the formula (Y) in the presence of an enzyme having prenyl transferase activity.
Herein, the term "in the presence of an enzyme having prenyl transferase activity" means in the presence of:
a culture of the above organism, an organism isolated from the culture, a treated product of the organism, or an enzyme purified from the culture or organism; or
A culture of an organism transformed by a genetic engineering technique (transformant) for expressing an enzyme having prenyl transferase activity, an organism isolated from the culture, a treated matter of the organism, or an enzyme purified from the culture or organism; or an analog thereof.
The skilled worker can prepare an organism transformed to express an enzyme having prenyl transferase activity by a conventionally known genetic engineering technique.
The organism of the above organism can be obtained by culturing the organism in an appropriate medium. These media are not particularly limited as long as they can proliferate the organism, and may be any media containing usual carbon sources, nitrogen sources, inorganic ions, and organic nutrient sources added as necessary.
For example, the carbon source may be any carbon source that the above-mentioned organism can utilize. Specific examples thereof include sugars such as glucose, fructose, maltose, amylose and sucrose, alcohols such as sorbitol, ethanol and glycerol, organic acids such as fumaric acid, citric acid, acetic acid and propionic acid and salts thereof, carbohydrates such as paraffin and mixtures thereof.
Examples of the nitrogen source include inorganic ammonium 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, organic nitrogen compounds such as peptone, yeast extract, meat extract and corn steep liquor, and mixtures thereof.
Other nutrient sources used in a usual medium, for example, inorganic salts, trace metal salts, vitamins, hormones, etc. may be appropriately mixed and used.
The culture conditions are also not particularly limited. For example, under aerobic conditions, the culture can be carried out for about 12 to 480 hours while appropriately controlling the pH to be in the range of 5 to 8 and the temperature to be in the range of 10 to 60 ℃.
The culture of the organism may be, for example, a culture solution obtained by culturing the organism under the above-mentioned culture conditions, or a culture filtrate (culture supernatant) obtained by separating the organism (organism) from the culture solution by filtration or the like. Further, the organism separated from the above culture may be, for example, an organism (organism) obtained by separation from a culture solution by filtration, centrifugation or the like.
The treated material of the organism may be, for example, an organism homogenate obtained by homogenizing an organism separated from the above culture, or an organism homogenate obtained by subjecting an organism to ultrasonic treatment.
The enzyme purified from the culture or the organism is, for example, an enzyme obtained by subjecting an enzyme present in the culture or the organism to a known purification procedure such as salting out, ion exchange chromatography, affinity chromatography, or gel filtration chromatography. The purity of the purified enzyme is not particularly limited.
The isoprene oligomer of the present invention can be produced by reacting allyl diphosphate represented by the formula (X) with a compound represented by the formula (Y) in the presence of an enzyme having prenyl transferase activity. Specifically, for example, the reaction is carried out by adding a culture of the organism, a purified enzyme or the like to a solution containing allyl diphosphate represented by the formula (X) and a compound represented by the formula (Y). Further, the reaction temperature may be, for example, 20 to 60 ℃, the reaction time may be, for example, 1 to 16 hours, and the pH may be, for example, 5 to 8. Further, magnesium chloride, a surfactant, 2-mercaptoethanol, and other substances may also be added as required.
In the isoprene oligomer of the present invention produced by the above-described reaction, Y in the formula (Z-1), the formula (Z-2), the formula (Z-1-1) or the formula (Z-2-1) is usually OPP or a hydroxyl group. OPP is from IPP or R-IPP. OPP is also readily hydrolyzed, which produces hydroxyl groups. This is why Y in the formula (Z-1), (Z-2), (Z-1-1) or (Z-2-1) is usually OPP or hydroxy.
Further, the isoprene oligomer in which Y in the formula (Z-1) or (Z-2) is a formyl group can be produced, for example, by oxidizing an isoprene oligomer in which Y in the formula (Z-1) or (Z-2) is OPP.
Further, the isoprene oligomer in which Y in the formula (Z-1) or (Z-2) is a carboxyl group can be produced, for example, by oxidizing an isoprene oligomer in which Y in the formula (Z-1) or (Z-2) is OPP.
Further, the isoprene oligomer in which Y in the formula (Z-1) or (Z-2) is an alkoxycarboxyl group, the isoprene oligomer in which Y in the formula (Z-1) or (Z-2) is an alkoxycarbonyl group can be produced, for example, by carboxylating the isoprene oligomer in which Y in the formula (Z-1) or (Z-2) is OPP in the above-mentioned manner and further esterifying the carboxylated isoprene oligomer.
The isoprene oligomer of the present invention can be produced by biosynthesis, in addition to the organic synthesis of the compound represented by the formula (Y) and the starting substrate of the allyl diphosphate derivative which can be selected as needed. Therefore, the invention considers the problems of exhaustion of petroleum resources and environment.
(polyisoprene)
Next, the polyisoprene of the present invention will be described. The polyisoprene of the present invention is synthesized (biosynthesized) from the allyl diphosphate represented by the formula (X) and the compound represented by the formula (Y). The monomer polymerized to the allyl diphosphate represented by the formula (X) may be a combination of the compound represented by the formula (Y) and isopentenyl diphosphate. For example, the polyisoprene of the present invention can be synthesized (biosynthesized) from the isoprene oligomer of the present invention and the compound represented by the formula (Y) and/or isopentenyl diphosphate. For example, the polyisoprene of the present invention may be synthesized (biosynthesized) from an unmodified isoprene oligomer and a compound represented by the formula (Y), or from an unmodified isoprene oligomer, a compound represented by the formula (Y) and isopentenyl diphosphate.
The polyisoprene of the invention has a structure similar to that of natural rubber and is highly compatible with rubber molecules. The polyisoprenes according to the invention also have a modified molecular skeleton and can therefore interact strongly with fillers (for example silica). Therefore, since the polyisoprene of the present invention is highly compatible with rubber while being strongly interactive with a filler (e.g., silica), if the polyisoprene is used for a rubber composition, a rubber composition in which rubber molecules are mixed with a filler to a higher degree than in conventional compositions can be obtained. For example, the rubber composition can thereby enhance low heat build-up, wet grip performance, abrasion resistance, elongation at break, and tensile strength at break.
The backbone of the polyisoprene of the present invention comprises polar groups or analogues thereof. Thus, the dispersibility of the filler (e.g., silica) is higher than in the case where a polar group (e.g., terminal diphosphate group) or the like is present only at the chain end. Therefore, for example, the improvement effects of low heat build-up, wet grip performance, abrasion resistance, elongation at break and tensile strength at break are increased.
Further, when the allyl diphosphate represented by the formula (X) is an allyl diphosphate represented by the formula (X-1), and at least one atom or atom group contained in the moiety II in the formula (X-1) is substituted with another atom or atom group, and none of the atoms or atom groups contained in the moiety III in the formula (X-1) is substituted with another atom or atom group, polyisoprene in which not only the skeleton but also the chain end is modified can be obtained. Since the polyisoprene has not only a modified skeleton but also a modified terminal, it can better interact with a filler (e.g., silica), and if the polyisoprene is used for a rubber composition, a rubber composition in which rubber molecules are mixed with a filler to a higher degree than in a conventional composition can be obtained. For example, the rubber composition can thereby provide further enhanced low heat build-up, wet grip performance, abrasion resistance, elongation at break, and tensile strength at break.
The number of isoprene units added to the allyldiphosphonic acid starting substrate of formula (X) in the polyisoprene of the present invention is preferably from 30 to 40000, more preferably from 31 to 38000, further preferably from 1000 to 35000, particularly preferably from 2000 to 30000.
For example, a specific example of the polyisoprene of the present invention is a polyisoprene represented by formula (ZZ-1) or formula (ZZ-2), wherein at least one atom or group of atoms contained in the v moiety in formula (ZZ-1) or formula (ZZ-2) is substituted with another atom or group of atoms;
Figure BDA0001630113170000261
wherein n in the formula (ZZ-1) represents an integer of 1 to 10; q represents an integer of 30 to 40000; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP;
Figure BDA0001630113170000262
wherein n in the formula (ZZ-2) represents an integer of 1 to 10; q represents an integer of 30 to 40000; y represents a hydroxyl group, a formyl group, a carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or OPP.
The atom or group of atoms contained in moiety v in formula (ZZ-1) or formula (ZZ-2) and the other atoms or groups of atoms may be as described for formula (X-1).
The moiety iv in the formulae (ZZ-1) and (ZZ-2) is derived from the allyldiphosphonic acid starting substrate represented by the formula (X). Thus, n in the formulae (ZZ-1) and (ZZ-2) is defined in the same manner as n in the formula (X).
The moiety v in formula (ZZ-1) and (ZZ-2) consists of an isoprene unit added to the allyldiphosphonate starting substrate represented by formula (X). Thus, q in formula (ZZ-1) and (ZZ-2) corresponds to the number of isoprene units added to the allyldiphosphonate starting substrate of formula (X).
Y in the formulae (ZZ-1) and (ZZ-2) is preferably OPP, hydroxyl or carboxyl due to a strong interaction with a filler (e.g., silica).
As described above, the present invention is based on the following findings: if the structure other than the methyl group at the 3-position in the structure of isopentenyl diphosphate is maintained, it can be used to produce isoprene oligomers or polyisoprenes by naturally occurring enzymes even if the methyl group at the 3-position is substituted with a desired group. Accordingly, the polyisoprene represented by the formula (ZZ-1) or the formula (ZZ-2) is preferably polyisoprene represented by the formula (ZZ-1-1) or the formula (ZZ-2-1):
Figure BDA0001630113170000271
wherein n, q and Y in formula (ZZ-1) are defined in the same manner as n, q and Y in formula (ZZ-1), and R is defined in the same manner as R in formula (Y);
Figure BDA0001630113170000272
wherein n, q and Y in formula (ZZ-2-1) are defined in the same manner as n, q and Y in formula (ZZ-2), and R is defined in the same manner as R in formula (Y).
As described above, it is also preferable to modify not only the backbone but also the chain ends at the same time. Thus, it is preferred that at least one atom or group of atoms contained in moiety iv of formula (ZZ-1) or formula (ZZ-2) (preferably formula (ZZ-1-1) or formula (ZZ-2-1)) is replaced by another atom or group of atoms.
The atom or group of atoms contained in the moiety iv in formula (ZZ-1), formula (ZZ-2), formula (ZZ-1-1) or formula (ZZ-2-1) and the other atoms or groups of atoms may be as described for formula (X-1).
Further, as described above, it is preferable that the allyl diphosphate represented by the formula (X) is an allyl diphosphate represented by the formula (X-1), and that at least one atom or atomic group contained in the moiety II in the formula (X-1) is substituted with another atom or atomic group, and that none of the atoms or atomic groups contained in the moiety III in the formula (X-1) is substituted with another atom or atomic group. Accordingly, with respect to the moiety iv in the formula (ZZ-1), the formula (ZZ-2), the formula (ZZ-1-1) and the formula (ZZ-2-1) represented by the following formula (Z), it is preferable that at least one atom or atomic group contained in the moiety iv-II in the formula (Z) is substituted with another atom or atomic group, and that none of the atoms or atomic groups contained in the moiety iv-III in the formula (Z) is substituted with another atom or atomic group.
Figure BDA0001630113170000281
The atom or atomic group contained in the moiety iv-II or the moiety iv-III in the formula (Z) and the other atom or atomic group may be the atom or atomic group described in the formula (X-1).
Specific examples of the moiety iv in which the chain end is also modified may include structures represented by the above formulae (a) to(s). Among them, the structures of the formulae (b), (c), (d), (e), (f), (k), (l) and (r) are preferable, and the structures of the formulae (b) and (c) are more preferable because they have stronger interaction with the filler (e.g., silica) and can more effectively improve low heat buildup, wet grip performance, abrasion resistance, elongation at break and tensile strength at break.
(method for producing polyisoprene)
The polyisoprene of the present invention can be produced from the isoprene oligomer of the present invention and the compound represented by the formula (Y) (R-IPP) and/or isopentenyl diphosphate (IPP) by the method (a) for synthesizing (biosynthesizing) polyisoprene. The ratio of the monomers of R-IPP and IPP used may be suitably varied according to the objective physical properties of the polyisoprene obtained, wherein the percentage of one monomer may be 0. Alternatively, the polyisoprene of the present invention may be produced by the method (B) for synthesizing (biosynthesizing) polyisoprene from an unmodified isoprene oligomer and a compound represented by the formula (Y), or may be produced by the method (C) for synthesizing (biosynthesizing) polyisoprene from an unmodified isoprene oligomer, a compound represented by the formula (Y) and isopentenyl diphosphate. In addition, in these cases, the ratio between the R-IPP and IPP monomers used may be suitably changed depending on the objective physical properties of the polyisoprene obtained.
The polyisoprene of the present invention can be produced by biosynthesis, in addition to the organic synthesis of the compound represented by the formula (Y) and the starting substrate of the allyl diphosphate derivative which can be selected as required. Therefore, the invention considers the problems of exhaustion of petroleum resources and environment.
Conventionally, it has been known that a natural rubber latex (in particular, a natural rubber latex from hevea brasiliensis) contains an enzyme and a rubber elongation factor (for example, an enzyme capable of addition polymerization of isopentenyl diphosphate as described above) or the like, and has an activity of catalyzing a condensation reaction between an isoprene oligomer and isopentenyl diphosphate, and therefore, catalyzes a reaction of sequentially linking isopentenyl diphosphate and isoprene oligomer into a Z configuration (newly added isoprene unit is in a cis configuration) to produce polyisoprene, as shown below.
Figure BDA0001630113170000291
Meanwhile, it is known that natural rubber latex (rubber latex) derived from some plants contains an enzyme and a rubber elongation factor (for example, an enzyme capable of addition polymerization of isopentenyl diphosphate as described above) or the like, which have an activity of catalyzing a condensation reaction between an isoprene oligomer and isopentenyl diphosphate, and thus, catalyzes a reaction of sequentially linking isopentenyl diphosphate to an isoprene oligomer in an E configuration (newly added isoprene unit is in a trans configuration) to produce polyisoprene.
As described above, when the methyl group at the 3-position in the structure of isopentenyl diphosphate is substituted with a desired group and the structure of isopentenyl diphosphate other than the methyl group at the 3-position is maintained unchanged to obtain a compound of formula (Y) (R-IPP), polyisoprene can be formed by using the compound and employing a naturally occurring enzyme, rubber elongation factor, or the like having an activity of catalyzing the above-mentioned reaction. Therefore, in the present invention, polyisoprene can be produced using the above-mentioned enzyme, rubber elongation factor or the like.
In other words, the method (a) of synthesizing (biosynthesizing) the polyisoprene of the present invention from the isoprene oligomer of the present invention and the compound represented by the formula (Y) and/or isopentenyl diphosphate, the method (B) of synthesizing (biosynthesizing) the polyisoprene of the present invention from the unmodified isoprene oligomer and the compound represented by the formula (Y), and the method (C) of synthesizing (biosynthesizing) the polyisoprene of the present invention from the unmodified isoprene oligomer, the compound represented by the formula (Y) and isopentenyl diphosphate can be accomplished, for example, by using an enzyme, a rubber elongation factor or the like contained in a natural rubber latex. An enzyme cloned from natural rubber latex, rubber elongation factor or the like can also be used for the above method.
Specifically, in the method (a), the isoprene oligomer of the present invention may be reacted with the compound represented by the formula (Y) and/or isopentenyl diphosphate in the presence of an enzyme and/or a rubber elongation factor. Likewise, in the method (B), an unmodified isoprene oligomer may be reacted with the compound represented by the formula (Y) in the presence of an enzyme and/or a rubber elongation factor. Similarly, in the method (C), an unmodified isoprene oligomer may be reacted with the compound represented by the formula (Y), isopentenyl diphosphate, in the presence of an enzyme and/or a rubber elongation factor.
More specifically, for example, the reaction may be initiated by adding a natural rubber latex, or an enzyme, rubber elongation factor or the like isolated from a natural rubber latex, to a solution containing the isoprene oligomer of the present invention and the compound represented by formula (Y) and/or isopentenyl diphosphate. Further, the reaction temperature may be, for example, 10 to 60 ℃, the reaction time may be, for example, 1 to 72 hours, and the pH may be, for example, 6 to 8. Further, magnesium chloride, a surfactant, 2-mercaptoethanol, potassium fluoride, and others may be added as necessary. Even in the case of using an unmodified isoprene oligomer, the reaction can be carried out under the same conditions.
In the present invention, the isoprene chain of polyisoprene may be extended in any direction (trans configuration or cis configuration). In other words, for example, the polyisoprene of the invention may be a polyisoprene in which all isoprene units are linked in the trans configuration (e.g., an isoprene oligomer of formula (ZZ-1)), a polyisoprene in which the isoprene units are linked in the trans-cis configuration (e.g., a polyisoprene of formula (Z-2)), or a polyisoprene in which the isoprene units are linked in the trans-cis-trans configuration. Among them, polyisoprene in which isoprene units are linked in a trans-cis configuration is preferable because it has the same structure as natural rubber derived from hevea brasiliensis, which is widely used in industry.
In the polyisoprene of the present invention produced by the reaction described above, Y in the formula (ZZ-1), the formula (ZZ-2), the formula (ZZ-1-1) or the formula (ZZ-2-1) is usually OPP or a hydroxyl group. OPP is from IPP or R-IPP. OPP is also readily hydrolyzed, which produces hydroxyl groups. This is why Y in formula (ZZ-1), (ZZ-2), (ZZ-1-1), or (ZZ-2-1) is typically OPP or hydroxy.
The polyisoprene of formula (ZZ-1) or (ZZ-2) wherein Y is a formyl group can be produced, for example, by oxidizing polyisoprene of formula (ZZ-1) or (ZZ-2) wherein Y is OPP.
The polyisoprene of formula (ZZ-1) or (ZZ-2) wherein Y is a carboxyl group can be produced, for example, by oxidizing polyisoprene of formula (ZZ-1) or (ZZ-2) wherein Y is OPP.
Further, the polyisoprene of formula (ZZ-1) or (ZZ-2) wherein Y is an alkoxycarboxyl group, or the polyisoprene of formula (ZZ-1) or (ZZ-2) wherein Y is an alkoxycarbonyl group can be produced, for example, by carboxylating the polyisoprene of formula (ZZ-1) or (ZZ-2) wherein Y is OPP and further esterifying the carboxylated polyisoprene in the above-mentioned manner.
The source of the natural rubber latex is not particularly limited. Examples thereof include: hevea brasiliensis (Heveabrasiensis), hevea brasiliensis (Ficus elastica), Ficus benghaiensis (Ficus lyrata), Ficus auriculata (Ficus benjamina), Ficus religiosa (Ficus religiosa), Ficus benghalensis (Ficus benghansis), Lactarius multiceps (Lactarius vollemus), Sonchus oleraceus L, Taraxacum officinale (Taraxacum mongolignale), and Helianthus annuus (Helianthus annuus). Among them, hevea brasiliensis is preferable because the rubber produced therefrom has a high molecular weight and the latex has a high rubber content.
The natural rubber latex can be obtained, for example, by scratching a groove-like wound on the trunk of the hevea brasiliensis tree with a knife or the like (this process is called "tapping"), followed by recovering the natural rubber latex flowing out of the cut milk tube.
Examples of the enzyme or rubber elongation factor separated from the natural rubber latex include a skim latex (Serum), a bottom layer phase (bottom fraction), and a rubber phase (rubber fraction) separated by centrifuging the natural rubber latex. The skim, base phase, and rubber phase contain an enzyme, rubber elongation factor, or the like.
(rubber composition)
The rubber composition of the present invention contains 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 build-up, wet grip performance, abrasion resistance, elongation at break and tensile strength at break (particularly low heat build-up and abrasion resistance). The polyisoprene of the present invention can be used as a rubber component.
The content of the polyisoprene in the present invention is preferably 20% by mass or more, more preferably 40% by mass or more, and further preferably 60% by mass or more based on 100% by mass of the rubber component. It may be 100 mass%.
Examples of materials that can be used as the rubber component in addition to the polyisoprene of the present invention include: diene rubbers such as Isoprene Rubber (IR), Natural Rubber (NR), Butadiene Rubber (BR), Styrene Butadiene Rubber (SBR), Styrene Isoprene Butadiene Rubber (SIBR), Chloroprene Rubber (CR), and acrylonitrile butadiene rubber (NBR). These rubber materials may be used alone, or two or more of them may be used in combination. Among them, NR, BR and SBR are preferable.
When the isoprene oligomer of the present invention is used in a rubber composition, NR is preferably used as the rubber component for the reason of high compatibility with the isoprene oligomer. By using the isoprene oligomer of the present invention together with NR, the effects of the isoprene oligomer of the present invention can be more suitably obtained.
When the isoprene oligomer of the present invention is used in a rubber composition, the content of NR is preferably 20 mass% or more, more preferably 40 mass% or more, and further preferably 60 mass% or more based on 100 mass% of the rubber component. It may be 100 mass%.
The content of the isoprene oligomer of the present invention is preferably 1 part by mass or more, and more preferably 2 parts by mass or more, per 100 parts by mass of the rubber component. When the amount of the isoprene oligomer used is less than 1 part by mass, the effect may not be sufficiently exerted. The content of the isoprene oligomer is also preferably 20 parts by mass or less, and more preferably 15 parts by mass or less. Above the content of 20 parts by mass, strength and wear resistance may be reduced.
Examples of the filler used in the present invention include: silica, carbon black, clay and calcium carbonate.
The filler used in the present invention is preferably silica. When silica is used, the effects obtained by using the isoprene oligomer of the present invention and/or the polyisoprene of the present invention can be sufficiently obtained. Any silica may be used, and examples thereof include dry silica (anhydrous silicic acid), wet silica (hydrous silicic acid). Wet silica is preferred because it contains more silanol groups.
Carbon black is also preferably used as a filler in the present invention. In this case, the effects obtained by using the isoprene oligomer of the present invention and/or the polyisoprene of the present invention can be sufficiently obtained.
In addition to the above-mentioned components, other compounding agents commonly used in the production of rubber compositions, for example, silane coupling agents, zinc oxide, stearic acid, various antioxidants (i.e., antioxidants), softening agents (e.g., oils), waxes, vulcanizing agents (e.g., sulfur), and vulcanization accelerators may be appropriately contained in the rubber composition of the present invention.
The rubber composition of the present invention can be prepared by a conventionally known method. For example, the rubber composition can be produced by mixing the components using a rubber mixer such as an open roll, a banbury mixer, or the like, followed by vulcanization.
The rubber composition of the present invention can be suitably used for tire members (e.g., tread, sidewall, under tread, ply, breaker, and carcass) and the like.
(pneumatic tires)
The pneumatic tire of the present invention can be produced by a usual method using the rubber composition. Specifically, an unvulcanized rubber composition is extruded into a shape corresponding to a tire member (for example, a tread or a sidewall), then molded by a tire molding machine by a usual method, and then bonded to another tire member to form an unvulcanized tire, and the unvulcanized tire is hot-pressed in a vulcanizing machine to form a tire.
Examples
The present invention will be described in detail with reference to the following examples, but the present invention is not limited thereto.
(production example 1)
(Synthesis of 3-R-3-butenyl diphosphate (Compound (R-IPP) represented by formula (Y))
The target compound was synthesized using n-R-aldehyde as starting material. Exomethylene (a compound represented by the following formula (i)) was introduced in formic acid at the α -position of n-R-aldehyde using dimethylamine by the method of Green et al (m.b. Green, and w.j.hickinbotton, j.chem.soc.1957, 3262). Subsequently, the obtained compound was reduced to 2-R-allyl alcohol (a compound represented by the following formula (ii)) with lithium aluminum hydride. Further, this compound was chlorinated with phosphorus chloride in pyridine to 2-R-allyl chloride (a compound represented by the following formula (iii)), and then reacted with carbon dioxide in the presence of a freshly synthesized grignard reagent to obtain a carboxylic acid (a compound represented by the following formula (iv)). Then, this compound was reduced with lithium aluminum hydride to form an alcohol (a compound represented by the following formula (v)). Then, this compound was tosylated with tosyl chloride in pyridine (a compound represented by the following formula (vi)). Then, this compound was phosphorylated with trimethyl phosphate in acetonitrile, whereby a target product (a compound represented by the following formula (vii), that is, a compound represented by the formula (Y) (R-IPP)) was obtained. Intermediates and final products of each synthesis stage were confirmed by using TLC and instrumental analysis (IR and NMR).
When R is ethyl, propyl, butyl, phenyl, mercapto, hydroxyl, or amino, respectively, a compound (R-IPP) represented by formula (Y) is synthesized. When R is ethyl, propyl, butyl, phenyl, mercapto, hydroxyl and amino, R-IPP of formula (Y) is designated as R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively.
Figure BDA0001630113170000341
(example 1)
(production of isoprene oligomer (all-trans))
An isoprene oligomer in which all isoprene units are linked in the trans configuration is prepared as represented by the formula (Z-1-1-1).
(preparation of transformant)
First, a transformant is prepared. In preparation of the transformant, pET15b (pET15b/human-GGPS) into which human geranylgeranyl diphosphate synthase was introduced was used. The pET15b/human-GGPS is supplied by Professor Zhang Bo Shi (Association Professor Hiroshi SAGAMI, Institute of MultidisciplicationResearch for Advanced Materials, Tohoku University) of the Institute of Multiplex Materials science, northeast University.
Coli BL21(DE3) was transformed by heat shock method using pET15 b/human-GGPS. The transformant was plated on LB agar medium containing 50. mu.g/mL of ampicillin, and then cultured overnight at 37 ℃ to select a transformant.
(preparation of protein having prenyl transferase Activity)
The obtained E.coli BL21(DE3)/pET15b/human-GGPS (wild type) was inoculated into a test tube containing 3mL of LB medium containing 50. mu.g/mL of ampicillin, and cultured with shaking at 37 ℃ for 5 hours. To the resulting culture solution, 1mL of an aliquot was inoculated into a 500mL Erlenmeyer flask containing 100mL of LB medium containing 50. mu.g/mL of ampicillin, and the cells were cultured with shaking at 37 ℃ for 3 hours. Then, IPTG was added to a concentration of 0.1mmol/L, and the cells were cultured with shaking at 30 ℃ for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells were homogenized by ultrasonic waves, and then centrifuged to obtain a supernatant. From the supernatant, a protein having prenyl transferase activity was purified using HisTrap (Amersham). The purification of the protein was confirmed by SDS-PAGE.
(preparation of isoprene oligomer)
A reaction solution containing 10mg of the purified protein, 50mM Tris-HCl buffer (pH7.5), 40mM magnesium chloride, 25mM 2-mercaptoethanol, 1mM of a starting substrate (geranyl diphosphate (GPP), and 1mM isopentenyl diphosphate (IPP) or one of R-IPP produced in production example 1 was prepared, the reaction was carried out in a water bath at 37 ℃ for 1 hour, after the completion of the reaction, 100ml of saturated saline and 500ml of pentane were added, the mixture was stirred and then allowed to stand, then, the supernatant (pentane layer) was concentrated by evaporation to dryness, the residue portion was confirmed by NMR to confirm the product structure, which is an isoprene oligomer, and the concrete case of the isoprene oligomer thus obtained (n, m, and R in formula (Z-1-1-1) is shown in Table 1. here, based on the information of the starting substrate used and the isoprene chain length measured by TLC, n and m in the formula (Z-1-1-1) are calculated. Further, the R structure in the formula (Z-1-1-1) was identified by NMR, TLC and GC-MS.
Further, the isoprene oligomers obtained by using the monomers IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively, are referred to as an isoprene oligomer (t-control), an isoprene oligomer (t-A), an isoprene oligomer (t-B), an isoprene oligomer (t-C), an isoprene oligomer (t-D), an isoprene oligomer (t-E), an isoprene oligomer (t-F) and an isoprene oligomer (t-G), respectively, in the following experiments. In addition, in the experiment, isoprene oligomer (t-E) and isoprene oligomer (t-F) were added to the rubber composition as isoprene oligomer a and isoprene oligomer E, respectively.
[ Table 1]
Figure BDA0001630113170000361
(relative reactivity of monomers)
One of IPP or R-IPP in manufacturing example 1 was reacted with geranyl diphosphate (GPP) under the following conditions. The relative activity of each R-IPP for GPP is expressed as an index relative to the IPP activity (═ 100).
500ng of purified protein, 50mM Tris-HCl buffer (pH7.5), 40mM magnesium chloride, 25mM 2-mercaptoethanol, 12.5. mu.M GPP and 50. mu.M [1-14C]IPP or one of the R-IPPs prepared in manufacturing example 1. The reaction was carried out in a water bath at 37 ℃ for 1 hour. After the reaction, the activity under each condition was determined by liquid scintillation counting and TLC. The relative activities of each R-IPP with respect to the activity of IPP (═ 100) are shown in table 2.
The isoprene oligomer was purified in the same manner as in example 1 (preparation of isoprene oligomer). Next, details of the obtained isoprene oligomer (n, m and R in the formula (Z-1-1-1)) are shown in Table 2. Here, based on the information on the starting substrate used and the isoprene chain length measured by TLC, n and m in formula (Z-1-1-1) were calculated. Further, the R structure in the formula (Z-1-1-1) was identified by NMR, TLC and GC-MS.
[ Table 2]
Figure BDA0001630113170000362
The results of tables 1 and 2 show that even with R-IPP, the obtained isoprene oligomer has the same molecular weight as when IPP was used. It was also demonstrated that the backbone of the isoprene oligomer was modified in accordance with the R-IPP used.
(example 2)
(preparation of polyisoprene)
Polyisoprene was prepared using an isoprene oligomer in which all isoprene units are linked in the trans configuration as represented by formula (Z-1-1-1) (all trans).
A reaction solution containing 10. mu.L of the latex fraction, 50mM Tris-HCl buffer (pH7.5), 25mM magnesium chloride, 40mM 2-mercaptoethanol, 40mM potassium fluoride, 50. mu.M isoprene oligomer, and either 1mM IPP or R-IPP prepared in production example 1 was prepared. The reaction was carried out in a water bath at 30 ℃ for 3 days. After the reaction, the molecular weight was measured by GPC. Next, based on the numerical value of the 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.
The latex component used herein is skim latex obtained by ultracentrifugation of latex obtained from hevea brasiliensis.
The isoprene oligomer used was one of the 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) prepared in the above section (preparation of isoprene oligomer).
[ Table 3]
Figure BDA0001630113170000371
The results in Table 3 show that even with R-IPP, the polyisoprene obtained has the same molecular weight as when IPP is used. Further, the obtained polyisoprene was analyzed by NMR, TLC and GC-MS, and it was confirmed that the skeleton of polyisoprene was modified in accordance with the R-IPP used similarly to the case of isoprene oligomer.
(example 3)
(production of isoprene oligomer (trans-cis))
An isoprene oligomer in which isoprene units are linked in a trans-cis configuration is prepared as represented by the formula (Z-2-1-1).
Figure BDA0001630113170000381
(preparation of transformant)
First, a transformant is prepared. In preparation of the transformant, pET22B (pET22B/MLU-UPS) introduced with undecaprenyl diphosphate synthase of Micrococcus luteus B-P26 was used as a dsDNA template. The pET22b/MLU-UPS was provided by professor Jun of ancient mountain of the Institute of science of multicomponent Materials of northeast University (prof. Tanetoshi Koyama, Institute of Multidisciplicationresearch for Advanced Materials, Tohoku University).
E.coli BL21(DE3) was transformed by the heat shock method using pET22 b/MLU-UPS. The transformant was plated on LB agar medium containing 50. mu.g/mL of ampicillin, and then cultured overnight at 37 ℃ to select a transformant.
(preparation of protein having prenyl transferase Activity)
The obtained E.coli BL21(DE3)/pET22b/MLU-UPS was inoculated into a test tube containing 3mL of LB medium containing 50. mu.g/mL of ampicillin, and cultured with shaking at 37 ℃ for 5 hours. To the resulting culture solution, 1mL of an aliquot was inoculated into a 500mL Erlenmeyer flask containing 100mL of LB medium containing 50. mu.g/mL of ampicillin, and the cells were cultured with shaking at 37 ℃ for 3 hours. Then, IPTG was added to a concentration of 0.1mmol/L, and the cells were cultured with shaking at 30 ℃ for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells were homogenized by ultrasonic waves, and then centrifuged to obtain a supernatant. From the supernatant, a protein having prenyl transferase activity was purified using HisTrap (Amersham). The purification of the protein was confirmed by SDS-PAGE.
(preparation of isoprene oligomer)
A preparation containing 10mg of the purified protein, 50mM Tris-HCl buffer (pH7.5), 40mM magnesium chloride, 40mM Triton X-100, 25mM 2-mercaptoethanol, 1mM starting substrate (farnesyl diphosphate (FPP) and 1mM IPP or one of the R-IPPs prepared in production example 1. the reaction was carried out in a water bath at 37 ℃ for 1 hour, after completion of the reaction, 100ml of saturated saline and 500ml of 1-butanol were added, the mixture was stirred and then allowed to stand, then the supernatant (1-butanol layer) was concentrated by evaporation to dryness, the residue fraction was confirmed by NMR to confirm the product structure, which is an isoprene oligomer, and the concrete case of the obtained isoprene oligomer (n, m and R in formula (Z-2-1-1)) was as shown in Table 4, based on the information on the starting substrate used and the isoprene chain length by TLC, n and m in formula (Z-2-1-1) were calculated. Further, the R structure in the formula (Z-2-1-1) was identified by NMR, TLC and GC.
Further, isoprene oligomers obtained by using the monomers IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively, are referred to as an isoprene oligomer (tc-control), an isoprene oligomer (tc-A), an isoprene oligomer (tc-B), an isoprene oligomer (tc-C), an isoprene oligomer (tc-D), an isoprene oligomer (tc-E), an isoprene oligomer (tc-F) and an isoprene oligomer (tc-G), respectively, in the following experiments. In addition, in the experiment, isoprene oligomer (tc-E) was added as isoprene oligomer (b) to the rubber composition. In addition, in the experiment, each of the isoprene oligomer (tc-A), the isoprene oligomer (tc-B), the isoprene oligomer (tc-C), the isoprene oligomer (tc-D), the isoprene oligomer (tc-E), the isoprene oligomer (tc-F) and the isoprene oligomer (tc-G) was added to the rubber composition as an isoprene oligomer h, an isoprene oligomer i, an isoprene oligomer j, an isoprene oligomer k, an isoprene oligomer l, an isoprene oligomer m and an isoprene oligomer n, respectively.
[ Table 4]
Figure BDA0001630113170000391
(relative reactivity of monomers)
One of IPP or R-IPP in manufacturing example 1 was reacted with farnesyl diphosphate (FPP) under the following conditions. The relative activity of each R-IPP for FPP is expressed as an index relative to IPP activity (═ 100).
500ng of purified protein, 50mM Tris-HCl buffer (pH7.5), 40mM magnesium chloride, 40mM Triton X-100, 25mM 2-mercaptoethanol, 12.5. mu.M FPP and 50. mu.M [1-14C]IPP or one of the R-IPPs prepared in manufacturing example 1. The reaction was carried out in a water bath at 37 ℃ for 1 hour. After the reaction, the activity under each condition was determined by liquid scintillation counting and TLC. The relative activities of each R-IPP with respect to IPP activity (═ 100) are shown in table 5.
The isoprene oligomer was purified in the same manner as in example 3 section (preparation of isoprene oligomer). Next, details of the obtained isoprene oligomer (n, m and R in the formula (Z-2-1-1)) are shown in Table 5. Here, based on the information on the starting substrate used and the isoprene chain length measured by TLC, n and m in formula (Z-2-1-1) were calculated. Further, the R structure in the formula (Z-2-1-1) was identified by NMR, TLC and GC-MS.
[ Table 5]
Figure BDA0001630113170000401
The results of tables 4 and 5 show that even with R-IPP, the obtained isoprene oligomer has the same molecular weight as when IPP was used. It was also demonstrated that the backbone of the isoprene oligomer was modified in accordance with the R-IPP used.
(example 4)
(preparation of polyisoprene)
Polyisoprene is prepared using an isoprene oligomer in which isoprene units are linked in a trans-cis configuration as represented by formula (Z-2-1-1) (trans-cis).
A reaction solution containing 10. mu.L of the latex fraction, 50mM Tris-HCl buffer (pH7.5), 25mM magnesium chloride, 40mM 2-mercaptoethanol, 40mM potassium fluoride, 50. mu.M isoprene oligomer, and either 1mM IPP or R-IPP prepared in production example 1 was prepared. The reaction was carried out in a water bath at 30 ℃ for 3 days. After the reaction, the molecular weight was measured by GPC. Next, based on the numerical value of the molecular weight and information on the starting substrate used, the number of isoprene units added to the starting substrate FPP was calculated. The results are shown in table 6.
The latex component used herein is skim latex obtained by ultracentrifugation of latex obtained from hevea brasiliensis.
The isoprene oligomer used was one of the isoprene oligomer (tc-control), isoprene oligomer (tc-A), isoprene oligomer (tc-B), isoprene oligomer (tc-C), isoprene oligomer (tc-D), isoprene oligomer (tc-E), isoprene oligomer (tc-F) and isoprene oligomer (tc-G) prepared in the above section (preparation of isoprene oligomer). Further, in the subsequent experiment, polyisoprene produced using isoprene oligomer (tc-E), isoprene oligomer (tc-F) was added to the rubber composition as polyisoprene D and polyisoprene A, respectively. Furthermore, polyisoprene produced using the isoprene oligomer (tc-A), the isoprene oligomer (tc-B), the isoprene oligomer (tc-C), the isoprene oligomer (tc-D), the isoprene oligomer (tc-E), the isoprene oligomer (tc-F) and the isoprene oligomer (tc-G) was added to the rubber composition as polyisoprene I, polyisoprene J, polyisoprene K, polyisoprene L, polyisoprene M, polyisoprene N and polyisoprene P, respectively.
[ Table 6]
Figure BDA0001630113170000411
The results in Table 6 show that even with R-IPP, the polyisoprene obtained has the same molecular weight as when IPP is used. Further, the obtained polyisoprene was analyzed by NMR, TLC and GC-MS, and it was confirmed that the skeleton of polyisoprene was modified in accordance with the R-IPP used similarly to the case of isoprene oligomer.
(example 5)
(production of isoprene oligomer (all-trans))
An isoprene oligomer in which all isoprene units are linked in the trans configuration is prepared as represented by the formula (Z-1-1-1).
Figure BDA0001630113170000412
(preparation of transformant)
First, a transformant is prepared. For the preparation of the transformant, pET15b (pET15b/Bacillus-FPS) introduced with farnesyl diphosphate synthase from Bacillus stearothermophilus was used. The pET15b/Bacillus-FPS was provided by professor Jun ancient mountain Prof, Tanetoshi Koyama, Institute of MultidisciplicationResearch for Advanced Materials, Tohoku University, at the Institute of Multiplex Materials science, northeast University.
E.coli BL21(DE3) was transformed by heat shock method using pET15 b/bacillus-FPS. The transformant was plated on LB agar medium containing 50. mu.g/mL of ampicillin, and then cultured overnight at 37 ℃ to select a transformant.
(preparation of protein having prenyl transferase Activity)
The obtained E.coli BL21(DE3)/pET15b/Bacillus-FPS (wild type) was inoculated into a test tube containing 3mL of LB medium containing 50. mu.g/mL of ampicillin, and cultured with shaking at 37 ℃ for 5 hours. To the resulting culture solution, 1mL of an aliquot was inoculated into a 500mL Erlenmeyer flask containing 100mL of LB medium containing 50. mu.g/mL of ampicillin, and the cells were cultured with shaking at 37 ℃ for 3 hours. Then, IPTG was added to a concentration of 0.1mmol/L, and the cells were cultured with shaking at 30 ℃ for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells were homogenized by ultrasonic waves, and then centrifuged to obtain a supernatant. From the supernatant, a protein having prenyl transferase activity was purified using HisTrap (Amersham). The purification of the protein was confirmed by SDS-PAGE.
(preparation of isoprene oligomer)
A reaction solution containing 10mg of the purified protein, 50mM Tris-HCl buffer (pH7.5), 40mM magnesium chloride, 25mM 2-mercaptoethanol, 1mM of a starting substrate (dimethylallyl Diphosphate (DMAPP) and 1mM isopentenyl diphosphate (IPP) or one of the R-IPPs prepared in production example 1 was prepared, the reaction was carried out in a water bath at 37 ℃ for 1 hour, after the completion of the reaction, 100ml of a saturated saline solution and 500ml of pentane were added, the mixture was stirred and then allowed to stand, then, the supernatant (pentane layer) was concentrated to dryness by evaporation, the residue portion was confirmed by NMR to confirm the product structure, which is an isoprene oligomer, and the concrete case of the obtained isoprene oligomer (n, m and R in the formula (Z-1-1-1) is shown in Table 7, here, based on the information of the starting substrate used and the isoprene chain length measured by TLC, n and m in the formula (Z-1-1-1) are calculated. Further, the R structure in the formula (Z-1-1-1) was identified by NMR, TLC and GC-MS.
Further, isoprene oligomers obtained by using monomers IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively, hereinafter, referred to as isoprene oligomer (t-control-1), isoprene oligomer (t-A-1), isoprene oligomer (t-B-1), 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), respectively, in the experimental use.
[ Table 7]
Figure BDA0001630113170000431
(relative reactivity of monomers)
One of IPP or R-IPP prepared in manufacturing example 1 was reacted with dimethyl diphosphoric acid (DMAPP) under the following conditions. The relative activity of each R-IPP for DMAPP is expressed as an index relative to IPP activity (═ 100).
500ng of 50mM Tris-HCl buffer (pH7.5) containing the purified protein was prepared40mM magnesium chloride, 25mM 2-mercaptoethanol, 12.5. mu.M DMAPP and 50. mu.M of [1-14C]IPP or one of the R-IPPs prepared in manufacturing example 1. The reaction was carried out in a water bath at 37 ℃ for 1 hour. After the reaction, the activity under each condition was determined by liquid scintillation counting and TLC. The relative activities of each R-IPP with respect to IPP activity (═ 100) are shown in table 8.
The isoprene oligomer was purified in the same manner as in the section of example 5 (preparation of isoprene oligomer). Next, details of the obtained isoprene oligomer (n, m and R in the formula (Z-1-1-1)) are shown in Table 8. Here, based on the information on the starting substrate used and the isoprene chain length measured by TLC, n and m in formula (Z-1-1-1) were calculated. Further, the R structure in the formula (Z-1-1-1) was identified by NMR, TLC and GC-MS.
[ Table 8]
Figure BDA0001630113170000432
The results of tables 7 and 8 show that even with R-IPP, the obtained isoprene oligomer has the same molecular weight as when IPP is used. It was also demonstrated that the backbone of the isoprene oligomer was modified in accordance with the R-IPP used.
(example 6)
(preparation of polyisoprene)
Next, polyisoprene was prepared using an isoprene oligomer in which all isoprene units are linked in the trans configuration as represented by the formula (Z-1-1-1) (all trans).
A reaction solution containing 10. mu.L of the latex fraction, 50mM Tris-HCl buffer (pH7.5), 25mM magnesium chloride, 40mM 2-mercaptoethanol, 40mM potassium fluoride, 50. mu.M isoprene oligomer, and either 1mM IPP or R-IPP prepared in production example 1 was prepared. The reaction was carried out in a water bath at 30 ℃ for 3 days. After the reaction, the molecular weight was measured by GPC. Next, based on the numerical value of the molecular weight and information of the starting substrate used, the number of isoprene units added to the starting substrate DMAPP was calculated. The results are shown in Table 9.
The latex component used herein is skim latex obtained by ultracentrifugation of latex obtained from hevea brasiliensis.
The isoprene oligomer used was one of the isoprene oligomer (t-control-1), isoprene oligomer (t-A-1), isoprene oligomer (t-B-1), 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) prepared in the above section (preparation of isoprene oligomer).
[ Table 9]
Figure BDA0001630113170000441
The results in Table 9 show that even with R-IPP, the polyisoprene obtained has the same molecular weight as when IPP is used. Further, the obtained polyisoprene was analyzed by NMR, TLC and GC-MS, and it was confirmed that the skeleton of polyisoprene was modified in accordance with the R-IPP used similarly to the case of isoprene oligomer.
(example 7)
(production of isoprene oligomer (trans-cis))
Next, using an allyl diphosphate derivative, an isoprene oligomer (backbone-and chain end-modified isoprene oligomer) in which isoprene units represented by the formula (Z-2-1-1) are linked in the trans-cis configuration and in which not only the backbone but also the chain ends are modified, is prepared.
Figure BDA0001630113170000451
(preparation of allyl diphosphate derivative)
First, an allyl diphosphate derivative was synthesized.
(production example 2)
(Synthesis of 8-methoxy-3, 7-dimethyl-dodeca- (2E,6E) -dienyldiphosphoric acid (Compound represented by the formula (B))
Geraniol was used as the starting material for the synthesis of the target compound. Geraniol was acetylated with pyridine and acetic anhydride in anhydrous dichloromethane to obtain an acetate (a compound represented by the following formula (bi)) (yield 95%). Subsequently, carbon at the 8-position of the acetate was subjected to seleno-oxidation in ethanol to obtain an aldehyde (a compound represented by the following formula (bii)) (yield 24%). Subsequently, the aldehyde was subjected to alkaline hydrolysis with potassium hydroxide to form an alcohol (a compound represented by the following formula (biii)) (yield 38%). Subsequently, the alcohol was treated with imidazole or tert-butyldiphenylchlorosilane (TBDPS) in anhydrous dichloromethane to obtain a compound represented by the following (biv) (yield 80%). Then, this compound was reacted with butyl lithium in anhydrous ether to obtain butyl alcohol (a compound represented by the following formula (bv)) (yield 73%). Subsequently, this compound was converted into a sodium salt in anhydrous tetrahydrofuran, followed by addition of methyl iodide to the sodium salt and Williamson synthesis to obtain an ether compound (a compound represented by (bvi) below) (yield 95%). Then, this ether was subjected to elimination reaction using tetra-n-ammonium fluoride in anhydrous tetrahydrofuran to obtain an alcohol body (a compound represented by the following formula (bvii)) (yield 87%). Then, the primary hydroxyl group was substituted with chlorine using N-chlorosuccinimide and dimethyl sulfide in an anhydrous dichloromethane solvent at a temperature of-40 ℃ or lower to obtain a chloride (a compound represented by the following (bvii)) (yield 92%). Then, this chloride was diphosphorylated with tris (tetra-n-butylammonium) hydrogendiphosphoric acid in anhydrous acetonitrile to obtain the objective product (compound represented by the following formula (bix), i.e., compound represented by the formula (B)) (yield 26%).
Confirmation of intermediates and final products at each synthesis stage was performed using TLC and instrumental analysis (IR and NMR).
Figure BDA0001630113170000461
(production example 3)
(Synthesis of 8-hydroxy-3, 7-dimethyl-dodeca- (2E,6E) -dienyldiphosphoric acid (Compound represented by the formula (C))
Geraniol was used as the starting material for the synthesis of the target compound. Geraniol was acetylated with pyridine and acetic anhydride in anhydrous dichloromethane to obtain an acetate (a compound represented by the following formula (ci)) (yield 97%). Subsequently, carbon at the 8-position of the acetate was subjected to seleno-oxidation in ethanol to obtain an aldehyde (a compound represented by the following formula (cii)) (yield 20%). Subsequently, the aldehyde was subjected to alkaline hydrolysis with potassium hydroxide to form an alcohol (a compound represented by the following formula (ciii)) (yield 42%). Subsequently, the alcohol was treated with imidazole or tert-butyldiphenylchlorosilane (TBDPS) in anhydrous dichloromethane to obtain the compound represented by the following (civ) (yield 80%). Then, this compound was reacted with butyllithium in anhydrous ether to obtain butanol (yield 62%). Then, this pure product was subjected to elimination reaction using tetra-n-ammonium fluoride in anhydrous tetrahydrofuran to obtain a diol (a compound represented by the following formula (cvi)) (yield 94%). Then, the primary hydroxyl group was substituted with chlorine using N-chlorosuccinimide and dimethyl sulfide in an anhydrous methylene chloride solvent at-40 ℃ or lower to obtain a chloride (a compound represented by the following (cvii)) (yield 90%). Then, this chloride was diphosphorylated with tris (tetra-n-butylammonium) hydrogendiphosphoric acid in anhydrous acetonitrile to obtain the target product (the compound represented by the following formula (cviii), i.e., the compound represented by the formula (C)) (yield 46%).
Confirmation of intermediates and final products at each synthesis stage was performed using TLC and instrumental analysis (IR and NMR).
Figure BDA0001630113170000471
(preparation of mutant enzyme)
Using an allyl diphosphate derivative as a starting substrate, the reaction can be carried out to produce an isoprene oligomer even when a wild-type enzyme having prenyl transferase activity is used. However, in order to increase the reaction efficiency, a variant enzyme that increases the enzymatic activity of the allyldiphosphate derivative was prepared.
The reagents used were from the QuickChange Site-directed mutagenesis Kit (QuickChange Site-directed mutagenesis Kit) (Stratagene). The primers are designed to enable introduction of the mutation at the target site. Primers for mutagenesis were purchased from institute of medicinal and BIOLOGICAL research (MEDICAL & BIOLOGICAL LABORATORIES CO., LTD) (manufacturer: IDT). The primers were designed as follows.
Preparation of primers for the variant enzyme N77A
Forward primer 5'-act gaa gca tgg tct cgt cct aaa g-3' (SEQ ID NO: 1)
Reverse primer 5'-gag acc atg ctt cag ttg aaa atg c-3' (SEQ ID NO: 2)
Preparation of primers for the variant enzyme L91D
Forward primer 5'-gat gaa aga tcc ggg tga ttt ttt aa-3' (SEQ ID NO: 3)
Reverse primer 5'-cac ccg gat ctt tca tca agt aat ta-3' (SEQ ID NO: 4)
As the dsDNA template, pET22B (pET22B/MLU-UPS) into which undecaprenyl diphosphate synthase (hereinafter, also referred to as wild-type enzyme) derived from Micrococcus luteus B-P26 was introduced was used. The pET22b/MLU-UPS was provided by professor Jun of ancient mountain of the Institute of science of multicomponent Materials of northeast University (prof. Tanetoshi Koyama, Institute of Multidisciplicationaryresearch for Advanced Materials, Tohoku University). Mix 10 Xpfu polymerase buffer 2. mu. L, dsDNA template 2-20ng, forward primer 50ng, reverse primer 50ng, dNTPs 0.4. mu.L (each 2.5mM), ddH2O20. mu. L, Pfu polymerase (2.5U/. mu.l) in total, 0.4mL, was used for PCR. The conditions for the PCR reaction were as follows:
at 95 ℃, 30 seconds, one cycle;
95 ℃, 30 sec/55 ℃, 1 min/68 ℃, 8 min, 15 cycles.
After PCR, 0.4. mu.L of Dpn I was added to the PCR solution, and the PCR solution was treated with Dpn I at 37 ℃ for 1 hour. E.coli DH5 α was transformed by heat shock method using 1 to 10 μ L of Dpn I treatment fluid. The transformant was plated on LB agar medium containing 50. mu.g/mL of ampicillin, and then cultured overnight at 37 ℃ to select a transformant. Subsequently, the transformant was cultured overnight in LB medium containing 50. mu.g/mL of ampicillin, and a plasmid was prepared from the resulting culture solution by the alkali SDS method. The mutation of the plasmid was confirmed using a sequence analyzer.
(preparation of protein having prenyl transferase Activity)
The obtained E.coli BL21(DE3)/pET22b/MLU-UPS (wild type and mutant type) was inoculated into a test tube containing 3mL of LB medium containing 50. mu.g/mL of ampicillin, and cultured with shaking at 37 ℃ for 5 hours. To the resulting culture solution, 1mL of an aliquot was inoculated into a 500mL Erlenmeyer flask containing 100mL of LB medium containing 50. mu.g/mL of ampicillin, and the cells were cultured with shaking at 37 ℃ for 3 hours. Then, IPTG was added to a concentration of 0.1mmol/L, and the cells were cultured with shaking at 30 ℃ for 18 hours. The culture solution was centrifuged to obtain wet cells. The wet cells were homogenized by ultrasonic waves, and then centrifuged to obtain a supernatant. From the supernatant, a protein having prenyl transferase activity was purified using HisTrap (Amersham). The purification of the protein was confirmed by SDS-PAGE. The amino acid sequences of the variant enzyme N77A and the variant enzyme L91D thus obtained are as shown in SEQ ID NO: 5 and 6.
(preparation of isoprene oligomer)
A reaction solution containing 10mg of the purified protein, 50mM Tris-HCl buffer (pH7.5), 40mM magnesium chloride, 40mM Triton X-100, 25mM 2-mercaptoethanol, 1mM of a starting substrate (farnesyl diphosphate (FPP) or one of the allyl diphosphate derivatives prepared in production examples 2 and 3, and 1mM IPP or one of the R-IPP prepared in production example 1 was prepared, the reaction was carried out in a water bath at 37 ℃ for 1 hour, after the completion of the reaction, 100ml of saturated saline and 500ml of 1-butanol were added, the mixture was stirred and left to stand, then the supernatant (1-butanol layer) was concentrated by evaporation to dryness, the product structure was confirmed by NMR from the residue, and the product structure was isoprene oligomer was obtained m and R) are shown in Table 10. Here, based on the information on the starting substrate used and the isoprene chain length measured by TLC, n and m in formula (Z-2-1-1) were calculated. Further, the R structure in the formula (Z-2-1-1) was identified by NMR, TLC and GC-MS.
Further, isoprene oligomers obtained using the monomers IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively, using the compound represented by the formula (B) as a starting substrate with the variant enzyme N77A as an enzyme, hereinafter, referred to as isoprene oligomer (tc-control-AB), isoprene oligomer (tc-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) and isoprene oligomer (tc-G-AB), respectively, in the following experimental use.
Further, using the variant enzyme L91D as an enzyme and the compound represented by the formula (B) as a starting substrate, isoprene oligomers obtained using monomers IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively, hereinafter, referred to as 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), and isoprene oligomer (tc-G-DB), respectively, in the following experimental use.
Further, isoprene oligomers obtained using the monomers IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively, using the compound represented by the formula (C) as a starting substrate with the variant enzyme N77A as an enzyme, hereinafter, referred to as 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) and isoprene oligomer (tc-G-AC), respectively, in the following experimental use.
Further, using the variant enzyme L91D as an enzyme and a compound represented by the formula (C) as a starting substrate, isoprene oligomers obtained using monomers IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G, respectively, hereinafter, referred to as 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), respectively, in the following experimental use.
In addition, in the experiment, each of the isoprene oligomer (tc-E-AB), isoprene oligomer (tc-F-AB), isoprene oligomer (tc-E-DB), and isoprene oligomer (tc-F-DB) was added to the rubber composition as isoprene oligomer c, isoprene oligomer F, isoprene oligomer d, and isoprene oligomer g, respectively.
[ Table 10]
Figure BDA0001630113170000511
Figure BDA0001630113170000512
Figure BDA0001630113170000513
Figure BDA0001630113170000514
(relative reactivity of monomers)
One of IPP or R-IPP prepared in production example 1 and one of farnesyl diphosphate (FPP) or allyl diphosphate derivatives prepared in production examples 2 and 3 were reacted under the following conditions. The relative activities of each allyl diphosphate derivative and each R-IPP are expressed exponentially with respect to the activities of FPP and IPP, respectively, where the activities of FPP and IPP are set to 100.
Preparation of a purified protein containing 500ng, 50mM Tris-HCl buffer (pH7.5), 40mM magnesium chloride, 40mM Triton X-100, 25mM 2-mercaptoethanol, 12.5. mu.M FPP or one of the allyldiphosphoric acid derivatives prepared in preparation examples 2 and 3, and 50. mu.M [1-14C]IPP or one of the R-IPPs prepared in manufacturing example 1. The reaction was carried out in a water bath at 37 ℃ for 1 hour. After the reaction, the activity under each condition was determined by liquid scintillation counting and TLC. Allyl diphosphate derivatization with respect to IPP activity (═ 100)The relative activities of substance and R-IPP are shown in Table 11.
The isoprene oligomer was purified in the same manner as in example 7 (preparation of isoprene oligomer). Next, details of the obtained isoprene oligomer (n, m and R in the formula (Z-2-1-1)) are shown in Table 11. Here, based on the information on the starting substrate used and the isoprene chain length measured by TLC, n and m in formula (Z-2-1-1) were calculated. Further, the R structure in the formula (Z-2-1-1) was identified by NMR, TLC and GC-MS.
[ Table 11]
Figure BDA0001630113170000531
Figure BDA0001630113170000532
Figure BDA0001630113170000533
Figure BDA0001630113170000534
The results of tables 10 and 11 show that even when R-IPP and allyl diphosphate derivatives are used, the obtained isoprene oligomers have the same molecular weight as when IPP and FPP are used. It was also confirmed that the isoprene oligomer was obtained with modifications corresponding to the R-IPP and allyl diphosphate derivatives used.
(example 8)
(preparation of polyisoprene)
Next, polyisoprene was prepared using the following isoprene oligomers: prepared using an allyl diphosphate derivative, an isoprene oligomer in which isoprene units are linked in a trans-cis configuration and in which not only the backbone but also the chain ends are modified (backbone-and chain end-modified isoprene oligomer) as represented by formula (Z-2-1-1).
A reaction solution containing 10. mu.L of a latex fraction, 50mM Tris-HCl buffer (pH7.5), 25mM magnesium chloride, 40mM 2-mercaptoethanol, 40mM potassium fluoride, 50. mu.M isoprene oligomer, and either 1mM IPP or R-IPP prepared in production example 1 was prepared. The reaction was carried out in a water bath at 30 ℃ for 3 days. After the reaction, the molecular weight was measured by GPC. Next, based on the numerical value of the molecular weight and information of the starting substrate used, the number of isoprene units added to the starting substrate FPP or allyl diphosphate derivative was calculated. The results are shown in Table 12.
The latex component used herein is skim latex obtained by ultracentrifugation of latex obtained from hevea brasiliensis.
The isoprene oligomers used were isoprene oligomers (tc-control-AB), isoprene oligomers (tc-A-AB), isoprene oligomers (tc-B-AB), isoprene oligomers (tc-C-AB), isoprene oligomers (tc-D-AB), isoprene oligomers (tc-E-AB), isoprene oligomers (tc-F-AB), isoprene oligomers (tc-G-AB), isoprene oligomers (tc-control-DB), isoprene oligomers (tc-A-DB), isoprene oligomers (tc-B-DB), isoprene oligomers (tc-C-DB) prepared in the section above (preparation of isoprene oligomers), Isoprene oligomer (tc-D-DB), isoprene oligomer (tc-E-DB), isoprene oligomer (tc-F-DB), isoprene oligomer (tc-G-DB), 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), isoprene oligomer (tc-G-AC), isoprene oligomer (tc-control-DC), isoprene oligomer (tc-A-DC), One of 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).
In the experiments described later, polyisoprene produced using the isoprene oligomer (tc-E-AB), the isoprene oligomer (tc-F-AB), the isoprene oligomer (tc-E-DB), the isoprene oligomer (tc-F-DB), the isoprene oligomer (tc-E-AC) and the isoprene oligomer (tc-E-DC) was added to the rubber composition as polyisoprene E, polyisoprene B, polyisoprene F, polyisoprene C, polyisoprene G and polyisoprene H, respectively.
[ Table 12]
Figure BDA0001630113170000561
Figure BDA0001630113170000562
Figure BDA0001630113170000563
Figure BDA0001630113170000564
The results in Table 12 show that even with R-IPP and isoprene oligomer with modified backbone and chain ends, the obtained polyisoprene has the same molecular weight as when IPP and unmodified isoprene oligomer are used. Further, the obtained polyisoprene was analyzed by NMR, TLC and GC-MS, and it was confirmed that the polyisoprene was modified corresponding to the R-IPP and the isoprene oligomer modified at the skeleton and chain end used, similarly to the case of the isoprene oligomer.
The chain ends of the isoprene oligomers and polyisoprenes prepared in the examples (corresponding to Y of formula (Z-1), formula (Z-2), formula (ZZ-1) or formula (ZZ-2)) are hydroxyl groups or OPP.
The chemical reagents used in examples 9 to 41 and comparative examples 1 to 4 are listed below.
NR：TSR20
BR: BR01(JSR company)
Carbon black: DIABLACK (N220) (Mitsubishi chemical corporation)
Isoprene oligomers a to n: isoprene oligomers prepared in the above examples
Polyisoprenes A to P: polyisoprene prepared in the above examples
Zinc oxide: zinc oxide #1 (Mitsui Metal mining Co., Ltd.)
Stearic acid: stearic acid (Riyou Co.)
An anti-aging agent: NORAC 6C (N- (1, 3-dimethylbutyl) -N' -phenyl-p-phenylenediamine) (New chemical industries, Inc. in Dang Nei)
Wax: SUNNOC wax (Dai Xinxing chemical industry Co., Ltd.)
Sulfur: sulfur powder (Crane, chemical company)
Vulcanization accelerator NS: NOCCELER NS (N-tert-butyl-2-benzothiazolesulfenamide) (New chemical industries, Inc. in the interior)
Silicon dioxide: nipsil AQ (Wet process silica) (Nippon silica Co., Ltd.)
Silane coupling agent: si266 (bis (3-triethoxysilylpropyl) disulfide) (Texaco corporation)
Vulcanization accelerator DPG: NOCCELER D (N, N-diphenylguanidine) (New chemical industries, Inc. in Dang Nei)
Materials other than sulfur and a vulcanization accelerator were kneaded using a 1.7L banbury mixer according to the formulations shown in tables 13 to 16 to obtain kneaded materials. Subsequently, sulfur and a vulcanization accelerator were mixed with the kneaded mixture, and the mixture was kneaded with an open roll to obtain an unvulcanized rubber composition. Using a steam pressure vulcanizer at a pressure of 80kgf/cm2The unvulcanized rubber composition was vulcanized at 150 ℃ for 30 minutes to prepare a vulcanized rubber composition.
The vulcanized rubber composition thus prepared was subjected to the following evaluations. The results are shown in tables 13 to 16. The reference recipe in table 13 is the recipe of comparative example 1, the reference recipe in table 14 is the recipe of comparative example 2, the reference recipe in table 15 is the recipe of comparative example 3, and the reference recipe in table 16 is the recipe of comparative example 4.
(viscoelasticity test)
Tan was measured using a viscoelastometer (manufactured by Ikegaku corporation) under conditions of 70 ℃ and 2% strain (initial elongation). The value is expressed as an index, with the tan of the reference formulation being 100. The higher the index, the greater the heat generation. The formulation having an index of 100 or less is considered to improve heat build-up resistance (low heat build-up). In other words, the lower the index, the better the low heat build-up.
(Lambert-En abrasion test)
An abrasion test was carried out using a lambert abrasion tester (manufactured by wayobo corporation) under conditions of a load of 3kg, a slip ratio of 40% and a sand speed of 15 g/min. The sample had a shape of 5mm in thickness and 50mm in diameter. The grindstone used was a GC grind of particle size # 80. The test results are expressed as an index, with the tan of the reference formulation being 100 (reference). The higher the index, the better the abrasion resistance. Formulations with indices greater than 100 are considered to have improved abrasion resistance.
(tensile test)
Tensile tests were carried out according to JIS K6251 "determination method of tensile stress and strain characteristics of rubber, vulcanized rubber or thermoplastic rubber" using No. 3 dumbbell-shaped test pieces composed of the vulcanized rubber sheets described above to measure tensile strength at break (TB) (MPa) and elongation at break (EB) (%). When a rubber composition having an elongation at break of less than 480% is used for a large tire, rubber fragments are likely to be generated, and improvement is required. Further, since the low tensile strength at break causes tire breakage, it is necessary to prevent the decrease in tensile strength at break due to the change of the material.
Figure BDA0001630113170000591
Figure BDA0001630113170000601
Figure BDA0001630113170000611
Figure BDA0001630113170000621
Tables 13 and 15 show that examples using the isoprene oligomer of the present invention exhibit excellent low heat build-up, abrasion resistance and elongation at break.
Tables 14 and 16 show that examples using the polyisoprene of the present invention exhibit excellent low heat build-up, abrasion resistance and tensile strength at break.
(sequence listing Individual text)
SEQ ID NO: 1: preparation of Forward primer for variant enzyme N77A
SEQ ID NO: 2: preparation of reverse primer for variant enzyme N77A
SEQ ID NO: 3: preparation of the Forward primer for the variant enzyme L91D
SEQ ID NO: 4: preparation of reverse primer for variant enzyme L91D
SEQ ID NO: 5: amino acid sequence of variant enzyme N77A
SEQ ID NO: 6: amino acid sequence of variant enzyme L91D
Sequence listing
<110> Sumitomo RUBBER INDUSTRIES, Ltd (Sumitomo RUBBER INDUSTRIES, LTD.)
National UNIVERSITY of French mountain UNIVERSITY (YAMAGATA UNIVERSITY)
<120> isoprene oligomer and method for producing isoprene oligomer
<140>2013800452905
<141>2013-09-02
<150>JP2012-200884
<151>2012-09-12
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<170>SIPOSequenceListing 1.0
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<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>primer_bind
<223> preparation of Forward primer for variant enzyme N77A
<400>1
actgaagcat ggtctcgtcc taaag 25
<210>2
<211>25
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>primer_bind
<223> preparation of reverse primer for variant enzyme N77A
<400>2
gagaccatgc ttcagttgaa aatgc 25
<210>3
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>primer_bind
<223> preparation of Forward primer for variant enzyme L91D
<400>3
gatgaaagat ccgggtgatt ttttaa 26
<210>4
<211>26
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>primer_bind
<223> preparation of reverse primer for variant enzyme L91D
<400>4
cacccggatc tttcatcaag taatta 26
<210>5
<211>249
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>ACT_SITE
<223> amino acid sequence of variant enzyme N77A
<400>5
Met Phe Pro Ile Lys Lys Arg Lys Ala Ile Lys Asn Asn Asn Ile Asn
1 5 10 15
Ala Ala Gln Ile Pro Lys His Ile Ala Ile Ile Met Asp Gly Asn Gly
20 25 30
Arg Trp Ala Lys Gln Lys Lys Met Pro Arg Ile Lys Gly His Tyr Glu
35 40 45
Gly Met Gln Thr Val Lys Lys Ile Thr Arg Tyr Ala Ser Asp Leu Gly
50 55 60
Val Lys Tyr Leu Thr Leu Tyr Ala Phe Ser Thr Glu Ala Trp Ser Arg
65 70 75 80
Pro Lys Asp Glu Val Asn Tyr Leu Met Lys Leu Pro Gly Asp Phe Leu
85 90 95
Asn Thr Phe Leu Pro Glu Leu Ile Glu Lys Asn Val Lys Val Glu Thr
100 105 110
Ile Gly Phe Ile Asp Asp Leu Pro Asp His Thr Lys Lys Ala Val Leu
115 120 125
Glu Ala Lys Glu Lys Thr Lys His Asn Thr Gly Leu Thr Leu Val Phe
130 135 140
Ala Leu Asn Tyr Gly Gly Arg Lys Glu Ile Ile Ser Ala Val Gln Leu
145 150 155 160
Ile Ala Glu Arg Tyr Lys Ser Gly Glu Ile Ser Leu Asp Glu Ile Ser
165 170 175
Glu Thr His Phe Asn Glu Tyr Leu Phe Thr Ala Asn Met Pro Asp Pro
180 185 190
Glu Leu Leu Ile Arg Thr Ser Gly Glu Glu Arg Leu Ser Asn Phe Leu
195 200 205
Ile Trp Gln Cys Ser Tyr Ser Glu Phe Val Phe Ile Asp Glu Phe Trp
210 215 220
Pro Asp Phe Asn Glu Glu Ser Leu Ala Gln Cys Ile Ser Ile Tyr Gln
225 230 235 240
Asn Arg His Arg Arg Phe Gly Gly Leu
245
<210>6
<211>249
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<221>ACT_SITE
<223> amino acid sequence of variant enzyme L91D
<400>6
Met Phe Pro Ile Lys Lys Arg Lys Ala Ile Lys Asn Asn Asn Ile Asn
1 5 10 15
Ala Ala Gln Ile Pro Lys His Ile Ala Ile Ile Met Asp Gly Asn Gly
20 25 30
Arg Trp Ala Lys Gln Lys Lys Met Pro Arg Ile Lys Gly His Tyr Glu
35 40 45
Gly Met Gln Thr Val Lys Lys Ile Thr Arg Tyr Ala Ser Asp Leu Gly
50 55 60
Val Lys Tyr Leu Thr Leu Tyr Ala Phe Ser Thr Glu Ala Trp Ser Arg
65 70 75 80
Pro Lys Asp Glu Val Asn Tyr Leu Met Lys Leu Pro Gly Asp Phe Leu
85 90 95
Asn Thr Phe Leu Pro Glu Leu Ile Glu Lys Asn Val Lys Val Glu Thr
100 105 110
Ile Gly Phe Ile Asp Asp Leu Pro Asp His Thr Lys Lys Ala Val Leu
115 120 125
Glu Ala Lys Glu Lys Thr Lys His Asn Thr Gly Leu Thr Leu Val Phe
130 135 140
Ala Leu Asn Tyr Gly Gly Arg Lys Glu Ile Ile Ser Ala Val Gln Leu
145 150 155 160
Ile Ala Glu Arg Tyr Lys Ser Gly Glu Ile Ser Leu Asp Glu Ile Ser
165 170 175
Glu Thr His Phe Asn Glu Tyr Leu Phe Thr Ala Asn Met Pro Asp Pro
180 185 190
Glu Leu Leu Ile Arg Thr Ser Gly Glu Glu Arg Leu Ser Asn Phe Leu
195 200 205
Ile Trp Gln Cys Ser Tyr Ser Glu Phe Val Phe Ile Asp Glu Phe Trp
210 215 220
Pro Asp Phe Asn Glu Glu Ser Leu Ala Gln Cys Ile Ser Ile Tyr Gln
225 230 235 240
Asn Arg His Arg Arg Phe Gly Gly Leu
245

Claims (10)

1. An isoprene oligomer synthesized from an allyl diphosphate represented by the following formula (X) and a compound represented by the following formula (Y), wherein the number of isoprene units added to the allyl diphosphate is 1 to 30,
Figure FDA0002424781690000011
in the formula (X), n represents an integer of 1 to 10, and a part of isoprene units may be modified;
Figure FDA0002424781690000012
in the formula (Y), R represents a nitrogen-containing group, an oxygen-containing group, a sulfur-containing group or a silicon-containing group.
2. The isoprene oligomer according to claim 1, which is synthesized from allyl diphosphate represented by the formula (X), a compound represented by the formula (Y), and isopentenyl diphosphate.
3. The isoprene oligomer according to claim 1 or 2, wherein the allyl diphosphate represented by the formula (X) is an allyl diphosphate represented by the following formula (X-1), at least one atom or atomic group contained in the moiety II in the following formula (X-1) is substituted with a different atom or atomic group, and an atom or atomic group contained in the moiety III in the following formula (X-1) is not substituted with a different atom or atomic group,
Figure FDA0002424781690000013
in the formula (X-1), n represents an integer of 2 to 10.
4. The isoprene oligomer according to claim 1 or 2, wherein the synthesis is performed using an enzyme having prenyl transferase activity.
5. An isoprene oligomer represented by the following formula (Z-1) or represented by the following formula (Z-2), wherein at least one atom or atomic group contained in the moiety v in the following formula (Z-1) or the following formula (Z-2) is substituted with a nitrogen atom, an oxygen atom, a sulfur atom, a silicon atom, a nitrogen-containing group, an oxygen-containing group, a sulfur-containing group or a silicon-containing group,
Figure FDA0002424781690000021
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 carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or an OPP group; at least one atom or group of atoms contained in moiety iv may be substituted with a different atom or group of atoms;
Figure FDA0002424781690000022
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 carboxyl group, an alkoxycarboxyl group, an alkoxycarbonyl group or an OPP group; at least one atom or group of atoms contained in moiety iv may be substituted with a different atom or group of atoms.
6. The isoprene oligomer according to claim 5, wherein at least one atom or atom group contained in the moiety iv in the formula (Z-1) or the formula (Z-2) is substituted with a different atom or atom group.
7. A process for producing an isoprene oligomer, characterized in that the isoprene oligomer is synthesized from an allyl diphosphate represented by the following formula (X) and a compound represented by the following formula (Y), the number of isoprene units added to the allyl diphosphate is 1 to 30,
Figure FDA0002424781690000023
in the formula (X), n represents an integer of 1 to 10, and a part of isoprene units may be modified;
Figure FDA0002424781690000024
in the formula (Y), R represents a nitrogen-containing group, an oxygen-containing group, a sulfur-containing group or a silicon-containing group.
8. The method for producing an isoprene oligomer according to claim 7, wherein the isoprene oligomer is synthesized from the allyl diphosphate represented by the formula (X), the compound represented by the formula (Y), and isopentenyl diphosphate.
9. The method for producing an isoprene oligomer according to claim 7 or 8, wherein the allyl diphosphate represented by formula (X) is an allyl diphosphate represented by formula (X-1) below, at least one atom or atomic group contained in the moiety II in formula (X-1) below is substituted with a different atom or atomic group, and an atom or atomic group contained in the moiety III in formula (X-1) below is not substituted with a different atom or atomic group
Figure FDA0002424781690000031
In the formula (X-1), n represents an integer of 2 to 10.
10. The method for producing an isoprene oligomer according to claim 7 or 8, wherein the synthesis is performed using an enzyme having prenyl transferase activity.
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