CN108484663A - Isoprene oligomer and the method for manufacturing isoprene oligomer - Google Patents

Isoprene oligomer and the method for manufacturing isoprene oligomer Download PDF

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Publication number
CN108484663A
CN108484663A CN201810340783.6A CN201810340783A CN108484663A CN 108484663 A CN108484663 A CN 108484663A CN 201810340783 A CN201810340783 A CN 201810340783A CN 108484663 A CN108484663 A CN 108484663A
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formula
isoprene
isoprene oligomer
diphosphonic acid
represented
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CN108484663B (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

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Mechanical Engineering (AREA)
  • Compositions Of Macromolecular Compounds (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Tires In General (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Polyoxymethylene Polymers And Polymers With Carbon-To-Carbon Bonds (AREA)

Abstract

The object of the present invention is to provide isoprene oligomer and polyisoprene that a kind of molecular skeleton is modified.The present invention also aims to provide a kind of rubber composition comprising the isoprene oligomer and/or the polyisoprene, and a kind of includes the pneumatic tire for being formed by the rubber composition tyre element (for example, tyre surface or sidewall).The present invention relates to a kind of isoprene oligomer, the compound synthesis represented by allyl diphosphonic acid and following formula (Y) represented by lower formula (X).In formula (X), n indicates integer of 1 to 10.In formula (Y), R indicates the group other than methyl.

Description

Isoprene oligomer and the method for manufacturing isoprene oligomer
The application is the divisional application based on following Chinese patent application：
The original bill applying date：On 09 02nd, 2013
Original bill application number：201380045290.5(PCT/JP2013/073509)
Original bill application title：Isoprene oligomer, polyisoprene, the side for manufacturing isoprene oligomer and polyisoprene Method, rubber composition and pneumatic tire
Technical field
The present invention relates to isoprene oligomer, polyisoprene, manufacture isoprene oligomer and polyisoprene It method, the composition containing isoprene oligomer and/or polyisoprene rubber and is filled by what the rubber composition was formed Air filled tyre.
Background technology
For a long time, people are dedicated to providing except the property that rubber has originally the rubber production also with new property Product import the filler of different shapes or other components that different materials make according to purposes in rubber composition, needed for acquisition The property wanted.For example, in automobile tire, people attempt by importing silica or carbon black etc. in the rubber phase of organic matter Filler, to improve the properties such as wearability, low-heat-generation or wet grip performance.
It is conventionally used in mixed fillers etc. in the rubber phases such as rubber composition, modified rubber (modified dienopolymer) Method further improves low-heat-generation, wet grip performance etc. to increase the compatibility between these components.These modified rubbers By making the rubber molecule in rubber phase be reacted with the compound for example containing nitrogen-containing group, chlorine sulfenyl, and in rubber point It is introduced and the affinity functional group of filler (for example, referenced patent document 1 and 2) in son.
Also, it is known that isoprene oligomer and polyisoprene have the diphosphonic acid of high reaction activity in its chain end Base etc..Therefore, the method for modifying of traditional isoprene oligomer or polyisoprene is related to above-mentioned high reaction activity end Diphosphonic acid base is reacted with the compound containing functional group.As described above, traditionally isoprene oligomer and poly- isoamyl two Alkene is mainly modified in the end of molecule.On the other hand, in conventional methods where, by the diphosphonic acid base of high reaction activity end or The skeleton of the influence of its analog, isoprene oligomer and polyisoprene cannot be fully modified.In addition, traditionally, The modification of natural rubber is only capable of by carrying out chemistry to the latex collected from Para rubber tree (Hevea brasiliensis) It handles to complete.It is thus impossible to by analysis be confirmed whether strand it is practical be modified and replace have it is what kind of Functional group.
Citation list
Patent document
Patent document 1：JP 2000-001573 A
Patent document 2：JP 2000-001575 A
Invention content
Technical problem
Present invention aim to address the above problems, and provide isoprene oligomer that a kind of molecular skeleton is modified and poly- Isoprene.The present invention also aims to provide a kind of rubber comprising the isoprene oligomer and/or the polyisoprene Glue composition, and provide a kind of comprising the inflated wheel for forming tyre element (for example, tyre surface or sidewall) by the rubber composition Tire.
Solution to the problem
The present invention relates to a kind of isoprene oligomer, the allyl diphosphonic acid represented by formula (X) and formula (Y) institute table The compound synthesis shown：
Wherein, n indicates integer of 1 to 10；
Wherein, R indicates the group other than methyl.
It is preferred that allyl diphosphonic acid of the isoprene oligomer represented by the formula (X), the formula (Y) indicated Compound and prenyl diphosphate synthesis.
It is preferred that the allyl diphosphonic acid represented by the formula (X) is the allyl diphosphonic acid represented by formula (X-1)：
Wherein, n indicates integer of 1 to 10, and at least one atom or atomic group quilt contained in the parts II in formula (X-1) Other (i.e. different) atoms or atomic group replace, and atom or atomic group be not by it contained in the parts III in formula (X-1) His atom or atomic group substitution.
The synthesis is preferably carried out by using with the active enzyme of prenyltransferases.
The invention further relates to the isoprene oligomer represented by a kind of formula (Z-1) or represented by formula (Z-2), formulas (Z-1) Or at least one atom or atomic group contained in the parts v in formula (Z-2) are replaced by other atoms or atomic group.
Wherein, the n in formula (Z-1) indicates integer of 1 to 10；M indicates 1 to 30 integer；Y indicate hydroxyl, formoxyl, carboxyl, Alkoxycarbonyl, alkoxy carbonyl or OPP；
Wherein, the n in formula (Z-2) indicates integer of 1 to 10；M indicates 1 to 30 integer；Y indicate hydroxyl, formoxyl, carboxyl, Alkoxycarbonyl, alkoxy carbonyl or OPP.
At least one atom or atomic group are by other atoms contained in the parts iv in preferred formula (Z-1) or formula (Z-2) Or atomic group substitution.
The invention further relates to a kind of methods of manufacture isoprene oligomer comprising the allyl represented by formula (X) Compound synthesis isoprene oligomer represented by diphosphonic acid and formula (Y)：
Wherein, n indicates integer of 1 to 10；
Wherein, R indicates the group other than methyl.
In the method for manufacture isoprene oligomer, the preferably described isoprene oligomer is represented by the formula (X) Allyl diphosphonic acid, the formula (Y) indicate compound and prenyl diphosphate synthesis.
It is preferred that the allyl diphosphonic acid represented by the formula (X) is the allyl diphosphonic acid represented by formula (X-1)：
Wherein, n indicates integer of 1 to 10, and at least one atom or atomic group quilt contained in the parts II in formula (X-1) Other atoms or atomic group replace, and atom or atomic group be not by other atoms or original contained in the parts III in formula (X-1) The substitution of son group.
The synthesis is preferably carried out by using with the active enzyme of prenyltransferases.
The invention further relates to a kind of polyisoprene, and the allyl diphosphonic acid and formula (Y) represented by formula (X) are represented Compound synthesis：
Wherein, n indicates integer of 1 to 10；
Wherein, R indicates the group other than methyl.
It is preferred that the chemical combination that allyl diphosphonic acid of the polyisoprene represented by the formula (X), the formula (Y) indicate Object and prenyl diphosphate synthesis.
It is preferred that the allyl diphosphonic acid represented by the formula (X) is the allyl diphosphonic acid represented by formula (X-1)：
Wherein, n indicates integer of 1 to 10, and at least one atom or atomic group quilt contained in the parts II in formula (X-1) Other atoms or atomic group replace, and atom or atomic group be not by other atoms or original contained in the parts III in formula (X-1) The substitution of son group.
The invention further relates to a kind of polyisoprene, represented by the isoprene oligomer, at least one formula (Y) Compound and prenyl diphosphate synthesis.
The invention further relates to a kind of formula (ZZ-1) is represented or formula (ZZ-2) represented by polyisoprene, formula (ZZ-1) or At least one atom or atomic group contained in the parts v in formula (ZZ-2) are replaced by other atoms or atomic group,
Wherein, the n in formula (ZZ-1) indicates integer of 1 to 10；Q indicates 30 to 40000 integer；Y indicate hydroxyl, formoxyl, Carboxyl, alkoxycarbonyl, alkoxy carbonyl or OPP；
Wherein, the n in formula (ZZ-2) indicates integer of 1 to 10；Q indicates 30 to 40000 integer；Y indicate hydroxyl, formoxyl, Carboxyl, alkoxycarbonyl, alkoxy carbonyl or OPP.
At least one atom or atomic group contained in the parts iv in preferred formula (ZZ-1) or formula (ZZ-2) are by other originals Son or atomic group substitution.
The invention further relates to a kind of methods of manufacture polyisoprene comprising by the isoprene oligomer, at least Compound and prenyl diphosphate represented by a kind of formula (Y) synthesize the polyisoprene.
The invention further relates to a kind of rubber compositions, and it includes in the isoprene oligomer and the polyisoprene At least one.
The present invention relates to a kind of pneumatic tires formed by the rubber composition.
Invention Beneficial Effect
The isoprene oligomer of the present invention is the allyl diphosphonic acid represented by formula (X) and the change represented by formula (Y) Close object synthesis isoprene oligomer or its be the isoprene oligomer indicated by formula (Z-1) or formula (Z-2), wherein At least one atom or atomic group contained in the parts v in formula (Z-1) or formula (Z-2) are replaced by other atoms or atomic group. In addition, the polyisoprene of the present invention is closed by the allyl diphosphonic acid represented by formula (X) and the compound represented by formula (Y) At polyisoprene；Compound and/or iso-amylene either represented by above-mentioned isoprene oligomer, a kind of formula (Y) The polyisoprene of base diphosphonic acid synthesis；Polyisoprene either represented by formula (ZZ-1) or represented by formula (ZZ-2), At least one atom or atomic group contained in the parts v in formula (ZZ-1) or formula (ZZ-2) are taken by other atoms or atomic group Generation.Therefore, the molecule (rubber molecule) of isoprene oligomer of the invention and the polyisoprene of the present invention all has modification Skeleton, thus its there is with filler (such as silica) outstanding compatibility.Therefore, if it is the isoprene of the present invention is low Polymers and/or the present invention polyisoprene be used for rubber composition, obtain rubber composition in rubber molecule can with fill out Material height mixes.For example, the rubber composition therefore provided has outstanding low-heat-generation and wearability.Further, For example, if in the tyre element (for example, tyre surface or sidewall) that above-mentioned rubber composition is used for, can provide with outstanding The pneumatic tire of low-heat-generation and wearability.
In addition, the method for the manufacture polyisoprene of the method and present invention of the manufacture isoprene oligomer of the present invention, can divide Indescribably for the high reaction activity end impregnable skeleton of diphosphonic acid base fully modified isoprene oligomer and poly- isoamyl two Alkene.
Further, because isoprene oligomer provided by the present invention and polyisoprene use the change represented by formula (Y) It closes object to obtain, functional group or other groups with known structure, Ke Yiming is added in the compound represented by the formula (Y) Really identify the functional group (modified group) in isoprene oligomer or polyisoprene.
Specific implementation mode
In artificial synthesized (biosynthesis) of rubber molecule (polyisoprene), enzyme can be used (for example, isoprene Based transferase) act on starting material (for example, farnesyl diphosphonic acid (FPP)) and monomer (for example, prenyl diphosphate) In mixture, generating the addition polymerization on starting material has the isoprene oligomer of 1 to 30 isoprene unit.Then, These isoprene oligomer are further mixed with the latex component of the enzyme containing addition polymerizable prenyl diphosphate. It is known that as a result, being formed in connection on oligomer, there are many polyisoprene of polyisoprene units.
As described above, form rubber molecule must use native enzyme the step of continuously linking monomer on starting material The addition polymerization of catalysis.
This mean that the starting material of the synthesis of rubber molecule (polyisoprene) and monomer needs be can use enzyme into The substance of row catalysis reaction.Therefore, it may be used as the structure of the starting material and monomer of rubber molecule (polyisoprene) material It is restricted.Particularly because the limit caused by the enzyme of prenyltransferases and energy addition polymerization prenyl diphosphate System, monomer are defined as naturally occurring prenyl diphosphate.
Its result cause the structure design flexibility of the rubber molecule (polyisoprene) of artificial synthesized (biosynthesis) by Limitation, so that being difficult to provide the flexibility in enough MOLECULE DESIGNs, carrys out the performance not available for additional natural rubber.
For these reasons, in order to prepare the rubber molecule (polyisoprene) for being imported with functional group or its analog, For example, traditionally, as described above, people for example, by by the end diphosphonic acid group of high reaction activity or its analog with contain The compound of functional group is reacted, to cause to be modified.Particularly, the modification of natural rubber is only capable of by from Para rubber tree The latex that (Hevea brasiliensis) is collected is chemically treated to complete.It is thus impossible to which carrying out confirmation by analysis is No strand is practical, which to be modified and replace, what kind of functional group.
Conversely, being found the present invention is based on following：By using the prenyl diphosphate of partially modified structure as monomer, Isoprene oligomer or polyisoprene are manufactured, the isoprene oligomer with functionalization skeleton or poly- isoamyl can be manufactured Diene.Further, because isoprene oligomer provided by the present invention and polyisoprene use prenyl diphosphate It obtains, functional group or other groups with known structure is added in the prenyl diphosphate, can positively identify The functional group (modified group) in isoprene oligomer or polyisoprene.
Particularly, it is found the present invention is based on following：If maintaining the structure of naturally occurring monomer prenyl diphosphate In the positions 3- methyl other than structure it is constant, can also or even by required group come when replacing positioned at 3- methyl Using the monomer prenyltransferases of enzyme or naturally occurring energy addition are generated by being used as naturally occurring oligomer The polymer for polymerizeing prenyl diphosphate generates enzyme, manufactures isoprene oligomer or polyisoprene.Its reason is not complete Portion is verified, but the enzyme that can be presumably due to prenyltransferases and energy addition polymerization prenyl diphosphate is attracted to In structure except the methyl of the positions 3- of prenyl diphosphate monomer, while these enzymes are relatively unwise to the structure of 3- methyl Sense.
Based on these discoveries, isoprene oligomer and polyisoprene that skeleton has required property can be provided, Therefore isoprene oligomer and polyisoprene with additional functions can be provided.
More particularly, in naturally occurring biosynthesis, for example, prenyl diphosphate (IPP) can continuously be gathered Close on dimethylallyl diphosphate (DMAPP), thus generate geranyl diphosphonic acid (GPP), farnesyl diphosphonic acid (FPP), Geranyl geranyl diphosphate (GGPP), geranyl farnesyl diphosphonic acid (GFPP), as follows.
Scheme 1
Conversely, the R-IPP that present invention use is obtained with the methyl of the positions 3- of required group (- R) substitution IPP, with Instead of prenyl diphosphate (IPP).In this case, continuously R-IPP can be aggregated on dimethylallyl diphosphate, Thus the geranyl diphosphonic acid (R-GPP) of framework modification, farnesyl diphosphonic acid (R-FPP), the framework modification of framework modification are generated Geranyl geranyl diphosphate (R-GGPP), framework modification geranyl farnesyl diphosphonic acid (R-GFPP), as follows.
Scheme 2
Further, if R-IPP and IPP used simultaneously, in other words, R-IPP and IPP is used in combination, by R-IPP and IPP It is continuously aggregated on dimethylallyl diphosphate, the geranyl diphosphonic acid (R- of framework modification as follows can be generated GPP), geranyl geranyl diphosphate (R-GGPP), the skeleton of the farnesyl diphosphonic acid (R-FPP) of framework modification, framework modification Modified geranyl farnesyl diphosphonic acid (R-GFPP).Therefore, tool can be manufactured with the ratio after adjusting and with R-IPP and IPP Needed the isoprene oligomer of property.
Scheme 3
Herein, the modification of the skeleton of molecule (rubber molecule) means with required functional group substitution molecule (rubber point Son) structure (particularly, part corresponding with the 3- of IPP position methyl) in IPP in skeleton, or mean to use other Structure (structure other than methyl) replaces the structure in IPP in molecule (rubber molecule) skeleton (particularly, with IPP's The 3- corresponding parts of methyl).
(isoprene oligomer)
A kind of allyl diphosphonic acid of oligomer represented by formula (X) of the present invention and the compound (R-IPP) represented by formula (Y) It synthesizes (biosynthesis)：
Wherein, n indicates integer of 1 to 10；
Wherein, R indicates the group other than methyl.
Herein, term " OPP (OPP groups) " means diphosphonic acid group (group represented by following formula (A-1)), and it has There are 3 hydroxyls being bonded with phosphorus atoms.When its in aqueous solution when, dissociation occurs for some or all of hydroxyl (for example, OPP becomes For the group as represented by following formula (A-2)).The intension of term " OPP " herein includes that above-mentioned part or all of hydroxyl solves From group.
The present invention isoprene oligomer have the structure close with natural rubber, and with rubber molecule height phase Hold.In addition, because the isoprene oligomer of the present invention has modified skeletal, can be sent out with filler (for example, silica) Raw strong interaction.Therefore, because the isoprene oligomer of the present invention and rubber highly compatible, while can be with filler Strong interaction occurs for (for example, silica), if the isoprene oligomer is used for rubber composition, can obtain phase Than traditional composition, rubber composition that rubber molecule is mixed with filler higher degree.For example, thus the rubber composition may be used To enhance low-heat-generation, wet grip performance, wearability, elongation at break and fracture tensile strength.
The skeleton of the isoprene oligomer of the present invention includes polar group or its analog.Filler is (for example, two as a result, Silica) dispersion performance when being only present in chain end higher than polar group or its analog the case where.Thus, for example, low life The improvement increase of hot, wet grip performance, wearability, elongation at break and fracture tensile strength.
The isoprene oligomer of the present invention also has outstanding antimicrobial acivity.Speculate this is because the present invention's is different Pentadiene oligomer has the structure different from naturally occurring usual isoprene oligomer, therefore, has and for example inhibits thin The enzyme and coenzyme of bacterium inhibit nucleic acid synthesis, the synthesis for inhibiting cell membrane synthesis, inhibiting cytoplasma membrane, damage cell membrane and damage Cytoplasma membrane and other effects.
First, the allyl diphosphonic acid represented by formula (X) is illustrated：
Wherein, n indicates integer of 1 to 10.
In formula (X), n indicates integer of 1 to 10 (preferably 1 to 4, more preferably 1 to 3).
The example of allyl diphosphonic acid represented by formula (X) includes：Dimethylallyl diphosphate (DMAPP), geranyl Diphosphonic acid (GPP), farnesyl diphosphonic acid (FPP), geranyl geranyl diphosphate (GGPP), geranyl farnesyl diphosphonic acid (GFPP).Wherein, preferably DMAPP, GPP and FPP, because they can be as the bottom of a plurality of types of prenyltransferases Object.
In allyl diphosphonic acid represented by formula (X), diphosphonic acid group is bonded with isoprene unit.Formula (X) institute table The allyl diphosphonic acid shown can be derived by being changed the allyl diphosphonic acid of (modification) to part isoprene unit Object.
The present inventor just uses allyl diphosphonic acid derivative to manufacture isoprene oligomer as starting material or gather different The method of pentadiene proposed patent application (JP2012-036360A).The patent application is summarized as follows.
As described above, starting material and the monomer needs of the synthesis of rubber molecule (polyisoprene) are can be carried out with enzyme It is catalyzed the substance of reaction.Which has limited the knots of the starting material and monomer of the material that may be used as rubber molecule (polyisoprene) Structure.Particularly because limitation caused by the enzyme of manufacture oligomer, starting material are restricted to naturally occurring dimethallyl Base diphosphonic acid, geranyl diphosphonic acid, farnesyl diphosphonic acid, geranyl geranyl diphosphate and the like.
On the contrary, the present inventor through extensively the study found that by using by such as structure by partially modified two phosphorus of farnesyl Acid etc. is used as starting material, manufactures isoprene oligomer or polyisoprene, can manufacture the chain end with functionalization Isoprene oligomer or polyisoprene.
Particularly, the inventors discovered that, if maintaining the following formula in the starting materials such as naturally occurring farnesyl diphosphonic acid (I) structure of the parts I in can also be used as day even if the other parts other than the parts I introduce required structure So existing oligomer generates the prenyltransferases of enzyme to manufacture isoprene oligomer.Its reason is not visited all It is bright, but can be presumably due in the structure for the parts I that prenyltransferases are attracted in the formula (I) of starting material, Structure relative insensitivity of these enzymes to other parts simultaneously.
Based on these discoveries, the isoprene oligomer and poly- isoamyl two of the chain end with required property can be provided Alkene, it is, therefore, possible to provide while not weakening the script property of isoprene oligomer or polyisoprene, it is additional there are many The isoprene oligomer and polyisoprene of function.
Therefore, in the present invention, the prenyltransferases that enzyme is generated by using naturally occurring oligomer, by formula (Y) compound of (R-IPP) is aggregated on starting material, which is while the I part-structures of the formula that maintains (I) The allyl diphosphonic acid for the formula (X) being modified can obtain and be not only skeleton and chain end is (from allyl diphosphonic acid Structure division) the isoprene oligomer that is also modified.
In particular it is preferred to which the allyl diphosphonic acid represented by the above-mentioned formula (X) is two phosphorus of allyl represented by formula (X-1) Acid：
Wherein, n indicate integer of 1 to 10 (n in formula (X-1) by with it is same in formula (X) in a manner of be defined), and formula (X-1) at least one atom or atomic group contained in the parts II in are replaced by other atoms or atomic group, in formula (X-1) Atom or atomic group contained in the parts III are not replaced by other atoms or atomic group.
As a result, having manufactured a kind of not only isoprene oligomer with modified skeletal, also with modified chain end. Because the isoprene oligomer, can be with filler (for example, dioxy not only with modified skeletal, also with modified chain end SiClx) preferably interact, if the isoprene oligomer is used for rubber composition, the building rubber compound obtained Rubber molecule in object can compare traditional composition, can carry out higher degree with filler and mix.For example, rubber composition by This can provide the low-heat-generation further enhanced, wet grip performance, wearability, elongation at break and fracture tensile strength.
Herein, the modification of the end of the molecule (rubber molecule) is it is meant that molecule (rubber molecule) end comes from formula (X) the predetermined part of the allyl diphosphonic acid structure represented by is replaced by required functional group, or means It is tied by other the predetermined part for coming from the allyl diphosphonic acid structure represented by formula (X) of molecule (rubber molecule) end Structure replaces.
Atom or atomic group are (that is, the atom before substitution or original contained in the parts II or the parts III in formula (X-1) Son group) example include hydrogen atom, methyl, methylene, carbon atom and methine.
The example of other atoms includes nitrogen-atoms, oxygen atom, sulphur atom, silicon atom and carbon atom.Wherein, preferably nitrogen is former Son because it is with strong intermolecular force, therefore can have strong interaction with enzyme or cell membrane, also with filler (for example, Silica) there is strong interaction.
Other described atomic groups can be nitrogen-containing group, oxygen-containing group, sulfur-containing group, silicon-containing group, carbon-containing group or its Analog.Its example includes：Acetoxyl group, alkoxy (preferably C1-C3 alkoxies, more preferable methoxyl group), hydroxyl, aryl are (excellent Select phenyl), alkyl (preferably C1-C5 alkyl, more preferable ethyl and tertiary butyl), acetyl group, N- alkyl-acetylamino (wherein, alkane Base preferably has 1 to 5 carbon atom) and azido.
From the angle of anti-microbe ability, preferably N- alkyl-acetylamino (more preferable N- methyl-acetamidos and N- fourths Base-acetylamino) and azido, because nitrogen-atoms has strong intermolecular force, therefore can occur with enzyme or cell membrane Strong interaction.Because of the strong interaction of itself and filler (for example, silica), it is also preferred that alkoxy and hydroxyl.
The example for the allyl diphosphonic acid (allyl diphosphonic acid derivative) that part isoprene unit is modified includes following Compound represented by formula (A) to (S).Wherein, preferred formula (B), the compound of (C), (D), (E), (F), (K), (L) and (R), The compound of more preferable formula (B) and (C), because they have the stronger interaction with filler (for example, silica), and And it can more effectively improve low-heat-generation, wet grip performance, wearability, elongation at break and fracture tensile strength.Based on excellent Elegant anti-microbe ability, it is also preferred that the structure of the formula (G) to (Q), more preferable formula (K), (L) and (Q) structure.
Technical staff can according to the method disclosed in JP2012-036360A, for example, from dimethylallyl diphosphate, Geranyl diphosphonic acid, farnesyl diphosphonic acid, geranyl geranyl diphosphate, geraniol, farnesol, trans-Geranylgeraniol or its class Like object, the allyl diphosphonic acid derivative represented by formula (A) to (S).
Then, the compound (R-IPP) represented by formula (Y) is illustrated：
Wherein, R indicates the group other than methyl.
In formula (Y), R can be the arbitrary group other than methyl.Its example include nitrogen-containing group, oxygen-containing group, sulfur-containing group, Silicon-containing group and carbon-containing group (in addition to methyl).Preference includes acetoxyl group, alkoxy, hydroxyl, aryl, alkyl (except methyl Outside), acetyl group, N- acetyl group-acetylamino, azido, amino and sulfydryl.Wherein, preferably nitrogen-containing group, oxygen-containing group, contain Methylthio group, silicon-containing group and carbon-containing group (in addition to methyl), more preferable alkyl (particularly, the alkyl of C2-C6), aryl are (especially Ground, phenyl), sulfydryl, hydroxyl and amino, further preferred sulfydryl and hydroxyl, the reason is that they can more effectively improve it is low Heat generation, wet grip performance, elongation at break and fracture tensile strength.
Technical staff can be according to the method illustrated in such as embodiment, the compound represented by formula (Y).
The addition isoprene on the allyl diphosphonic acid starting material of formula (X) in the isoprene oligomer of the present invention The quantity of unit preferably 1 to 30, more preferable 1 to 20, further preferred 1 to 10.
For example, the concrete example of the isoprene oligomer of the present invention is the isoprene represented by formula (Z-1) or formula (Z-2) At least one atom or atomic group are by other atoms or atom contained in the parts v in oligomer, formula (Z-1) or formula (Z-2) Group's substitution.
Wherein, the n in formula (Z-1) indicates integer of 1 to 10；M indicates 1 to 30 integer；Y indicate hydroxyl, formoxyl, carboxyl, Alkoxycarbonyl, alkoxy carbonyl or OPP；
Wherein, the n in formula (Z-2) indicates integer of 1 to 10；M indicates 1 to 30 integer；Y indicate hydroxyl, formoxyl, carboxyl, Alkoxycarbonyl, alkoxy carbonyl or OPP.
Atom or atomic group and other described atoms or atomic group contained in the parts v in formula (Z-1) or formula (Z-2) Can be the atom or atomic group as described in formula (X-1).
The parts iv in formula (Z-1) and (Z-2) are the knots for coming from the allyl diphosphonic acid starting material represented by formula (X) Structure.Therefore, the n in formula (Z-1) and (Z-2) is defined in a manner of identical with the n in formula (X).
Allyl diphosphonic acid starting material of the parts v represented by adding to formula (X) in formula (Z-1) and (Z-2) it is different Pentadiene unit forms.Therefore, the m in formula (Z-1) and (Z-2) is originated with the above-mentioned allyl diphosphonic acid for adding to formula (X) The quantity of the isoprene unit of substrate is corresponding.
Strong interaction due to outstanding antimicrobial properties and with filler (for example, silica), formula (Z-1) The preferred OPP of Y, hydroxyl or carboxyl in (Z-2).
As described above, being found the present invention is based on following：If maintaining the methyl of the positions 3- in prenyl diphosphate structure Structure in addition is constant, even if 3- methyl are replaced by required group, it can also be used by naturally occurring enzyme, Manufacture isoprene oligomer or polyisoprene.Therefore, the isoprene oligomer represented by formula (Z-1) or formula (Z-2) is excellent It is selected as that formula (Z-1-1) is represented or the isoprene oligomer as represented by formula (Z-2-1)：
Wherein, n, m and Y in formula (Z-1-1) are defined in a manner of identical with n, m and Y in formula (Z-1), R with formula (Y) same way of the R in defines；
Wherein, n, m and Y in formula (Z-2-1) are defined in a manner of identical with n, m and Y in formula (Z-2), R with formula (Y) same way of the R in defines；
As described above, it is also preferred that the substance that skeleton and chain end are modified simultaneously.It is therefore preferable that formula (Z-1) or formula (Z-2) At least one atom contained by the parts iv or atomic group in (preferred formula (Z-1-1) or formula (Z-2-1)) is by other atoms or original The substitution of son group.
Atom or atomic group contained in the parts iv in formula (Z-1), formula (Z-2), formula (Z-1-1) or formula (Z-2-1) with And other described atoms or atomic group can be as described in formulas (X-1).
Further, as described above, it is preferred to which the allyl diphosphonic acid represented by formula (X) is the allyl represented by formula (X-1) Base diphosphonic acid, and at least one atom or atomic group contained in the parts II in formula (X-1) are by other atoms or atomic group Replace, atom or atomic group contained in the parts III in formula (X-1) are not replaced by other atoms or atomic group.Therefore, it closes The parts iv in formula (Z-1), formula (Z-2), formula (Z-1-1) and the formula (Z-2-1) as represented by following formula (Z), in preferred formula (Z) The parts iv-II in contained at least one atom or atomic group replaced by other atoms or atomic group, in formula (Z) Atom or atomic group contained in the parts iv-III are not replaced by other atoms or atomic group.
In formula (Z) iv-II part or the parts iv-III contained in atom or atomic group and other described atoms or Atomic group can be the atom or atomic group as described in formula (X-1).
The concrete example for the parts iv that chain end is also modified may include the structure as represented by following formula (a) to (s).Its In, the structure of preferred formula (b), (c), (d), (e), (f), (k), (l) and (r), the structure of more preferable formula (b) and (c), because it There is the stronger interaction with filler (for example, silica), and can more effectively improve low-heat-generation, wet grab Ground performance, wearability, elongation at break and fracture tensile strength.Due to outstanding anti-microbe ability, it is also preferred that formula (g) is extremely (q) structure, more preferable formula (k), (l) and (q) structure.
(method of manufacture isoprene oligomer)
The present invention allyl diphosphonic acid of the isoprene oligomer represented by formula (X) and the compound represented by formula (Y) into Row synthesis (biosynthesis).
As the allyl diphosphonic acid represented by formula (X) and the compound synthesis (biosynthesis) represented by formula (Y) One example of the method for isoprene oligomer of the present invention, including use with the active enzyme of prenyltransferases.It is special Not, in the presence of enzyme active with prenyltransferases, allyl diphosphonic acid represented by formula (X) with can be in alkene Compound reaction represented by (Y) of the monomer polymerizeing in propyl diphosphonic acid formula.Allyl diphosphonic acid represented by formula (X) and formula (Y) compound represented by can be the composition of individual compound or multiple compounds respectively.It is, for example, possible to use formula (Y) composition of the multiple compounds of the different formulas (Y) of R in is low to provide the isoprene with required property Polymers.
In the present invention, the monomer polymerizeing on the allyl diphosphonic acid represented by formula (X) can only be formula (Y) (R-IPP) Represented compound.In this case, can be with isoprene oligomer shown in fabrication scheme 2.Alternatively, polymerization is in formula (X) Monomer on represented allyl diphosphonic acid can be the compound and prenyl diphosphate represented by formula (Y) (R-IPP) (IPP) combination.In this case, can be with isoprene oligomer shown in fabrication scheme 3.
Herein, term means " with prenyltransferases activity " with catalysis allyl substrate (allyl two Phosphoric acid) and the condensation reaction of prenyl diphosphate to synthesize the new allyl diphosphonic acid of the isoprene unit with addition Active enzyme, therefore, can be catalyzed by prenyl diphosphate continuously with allyl substrate (allyl diphosphonic acid) link Reaction.
As described above, when the methyl for replacing the positions 3- in prenyl diphosphate structure with required group and maintaining iso-amylene The structure in addition to 3- methyl of base diphosphonic acid is constant and obtains the compound of formula (Y) (R-IPP), can be by using this Compound simultaneously uses such as with the naturally occurring enzyme manufacture isoprene oligomer of the active enzyme of prenyltransferases.
As with the active enzyme of prenyltransferases, it has been found that a plurality of types of enzymes.
The enzyme (isoprene unit of each new addition has cis-configuration) that Z- isoprene chains can be extended includes：Z- nine Prenyl diphosphate synthase (Ishii, K.et al., (1986) Biochem., J., 233,773.), 11 iso-amylene Diphosphate synthase (Takahashi, I. and Ogura, K., (1982) J.Biochem., 92,1527.；Keenman, M.V. and Allen, C.M., (1974) Arch.Biochem.Biophys., 161,375.), Z- farnesyl diphosphate synthases (Identification of a short(C-15)chain Z-isoprenyl diphosphate synthase and a homologous long(C-50)chain isoprenyl diphosphate synthase in Mycobacterium Tuberculosis (short chain (C-15) the farnesyl diphosphate synthase in tubercle bacillus and homologous long-chain (C-50) isoamyl two The identification of alkene diphosphate synthase), Schulbach, MC., et al.JOURNAL OF BIOLOGICAL CHEMISTRY, 275 (30), (2000) 22876-22881) and the long alcohol radical diphosphate synthase of dehydrogenation ((Identification of human The knowledge of the long alcohol radical diphosphate synthase gene of dehydrodolichyl diphosphate synthase gene mankind's dehydrogenations Not), Endo, Shota.et al, Biochimica et Biophysica Acta (BBA), 1625 (3), (2003) p.291- 295.)。
Furthermore it is possible to extend enzyme (isoprene unit of each new addition has anti-configuration) packet of E- isoprene chains It includes：Farnesyl diphosphate synthase, geranyl geranyl diphosphate synzyme, six isoprene diphosphate synthases, seven isoamyls Diene diphosphate synthase, eight isoprene diphosphate synthases, ten isoprene diphosphate synthases.
The maximum number for the isoprene unit that can be formed and direction (anti-configuration or the cis- structure of isoprene chain elongation Type) depend on specific enzyme.Therefore, it can be selected according to the target numbers of isoprene unit and isoprene chain elongation direction Select enzyme.In the present invention, the isoprene chain of isoprene oligomer upper in any direction can extend (anti-configuration or cis- structure Type).In other words, for example, the isoprene oligomer of the present invention can be that all isoprene units are linked with anti-configuration Isoprene oligomer (for example, isoprene oligomer of formula (Z-1)), isoprene unit connects with trans-cis configuration The isoprene oligomer (for example, isoprene oligomer of formula (Z-2)) or isoprene unit of knot are with Trans-cis- The isoprene oligomer of anti-configuration connection.
Each tellurian existing biology is provided with the active enzyme of prenyltransferases.Have and carries isoamyl The biology of the enzyme of dialkylene transferase active it can be cited for example that, micrococcus luteus B-P26 (Micrococcus luteus B- P26), Escherichia coli (Escherichia coli), saccharomyces cerevisiae (Saccharomyces cerevisiae), arabidopsis (Arabidopsis thaliana), Para rubber tree (Hevea brasiliensis), periploca sepium (Periploca Sepium), bacillus stearothermophilus (Bachillus Stearothermophilus), sulfolobus acidocaldarius (Sulfolobus acidocaldarius (ATCC49426)), homo sapiens (Homo sapiens), sonchus oleraceus (Sonchus Oleracers L.), Western dandelion (Taraxacum officinale) and sunflower (Helianthus annuus).
Script substrate (starting material) with the active enzyme of prenyltransferases is allyl diphosphonic acid.Then, Because of inhibitor of the allyl diphosphonic acid derivative script as the enzyme, in many cases, if by allyl diphosphonic acid Derivative is used as starting material, relatively low (particularly, the formula of enzymatic activity which shows on the allyl diphosphonic acid derivative Compound represented by (G) to (Q)).For these reasons, when use allyl diphosphonic acid derivative as the feelings of starting material Under condition, the variant enzyme that can improve enzymatic activity is used for allyl diphosphonic acid derivative.It, can using variant enzyme To prepare the biology (transformant) converted by technique for gene engineering, to express variant enzyme.Particularly, technical staff can root According to the method disclosed in JP2012-036360A, the variant for the enzymatic activity for improving allyl diphosphonic acid derivative is easily prepared Enzyme.
The isoprene oligomer of the present invention can pass through in the presence of enzyme active with prenyltransferases Allyl diphosphonic acid represented by formula (X) reacts with the compound represented by formula (Y) and is prepared.
Herein, term means the case where there are following substances " in the presence of enzyme active with prenyltransferases " Under：
The culture of above-mentioned biology, the organism detached from the culture, the processed material of the organism, or from the training Support object or the enzyme of organism purifying；Or
For expressing the biology (conversion converted by technique for gene engineering with the active enzyme of prenyltransferases Son) culture, the organism detached from the culture, the processed material of the organism, or from the described culture or The enzyme of organism purifying；Or its analog.
Technical staff can be by known technique for gene engineering, and preparing for expressing there are prenyltransferases to live The enzyme of property and the biology being converted.
The biology, can be by being cultivated acquisition by the organism of above-mentioned biology in culture medium appropriate.These culture mediums As long as can make the biology be proliferated, be not particularly limited, can be containing common carbon source, nitrogen source, inorganic ions and The organic nutrition source being added as needed on is generally incubated base.
Can be the arbitrary carbon source that above-mentioned biology can utilize for example, as carbon source.Its specific example can make With the carbohydrate of glucose, fructose, maltose, amylose and sucrose etc., the alcohols of D-sorbite, ethyl alcohol, glycerine etc., rich horse The organic acid of acid, citric acid, acetic acid, propionic acid etc. and their salt, the carbohydrate of paraffin etc. and theirs is mixed Close object.
As the inorganic ammonium salt of the example of nitrogen source, including ammonium sulfate, ammonium chloride etc., ammonium fumarate, ammonium citrate etc. have The ammonium salt of machine acid, the nitrate of sodium nitrate, potassium nitrate etc., the organonitrogen compound of peptone, yeast extract, meat extract, corn steep liquor etc., And their mixture.
It can properly mix and use the other nutrient sources being generally incubated used in base, for example, inorganic salts, micro gold Belong to salt, vitamins, hormone etc..
Condition of culture is also not particularly limited.For example, under aerobic conditions, the model that can be 5 to 8 in suitable control pH It encloses, in the case of the range that temperature is 10 to 60 DEG C, carries out culture in 12 to 480 hours or so.
The culture of the biology can be for example, the training obtained by the biology of above-mentioned condition of culture culture Nutrient solution, or culturing filtrate (culture supernatant) that detach biological (organism) from the culture solution by filtering etc. by obtains.This Outside, the organism detached from above-mentioned culture can be obtained by for example, being detached from culture solution by filtering or centrifuging etc. The organism (biology) obtained.
The processed material of the organism can be for example, by being obtained after the organism homogenizing processing detached in above-mentioned culture The organism homogenate obtained, or the organism obtained after ultrasonication is carried out to biology and is homogenized.
The enzyme purified from the culture or the organism is for example, to existing in the culture or the organism Enzyme saltoutd, enzyme obtained from purification process well known to ion-exchange chromatography, affinity chromatography or gel filtration chromatography etc..It is pure The purity for changing enzyme is not particularly limited.
The isoprene oligomer of the present invention can pass through in the presence of enzyme active with prenyltransferases Allyl diphosphonic acid represented by formula (X) reacts with the compound represented by formula (Y) and is prepared.Specifically, for example, this is anti- It should be by adding the life in containing solution of the allyl diphosphonic acid with the compound represented by formula (Y) represented by formula (X) The culture of object, the enzyme of purifying or its analog carry out.In addition, reaction temperature can be such as 20 to 60 DEG C, the reaction time can Be such as 1 to 16 hour, pH can be such as 5 to 8.In addition it is also possible to as needed, addition magnesium chloride, surfactant, 2 mercapto ethanol and other substances.
In the isoprene oligomer of the present invention by the reaction manufacture of above description, formula (Z-1), formula (Z-2), formula (Z-1-1), the Y in formula (Z-2-1) is usually OPP or hydroxyl.OPP comes from IPP or R-IPP.OPP can also be hydrolyzed easily, OPP's Hydrolysis generates hydroxyl.This is why the Y in formula (Z-1), (Z-2), (Z-1-1) or (Z-2-1) is usually OPP or hydroxyl.
In addition, the Y in formula (Z-1) or (Z-2) is the isoprene oligomer of formoxyl, for example, can pass through oxidation-type (Z-1) Or the Y in (Z-2) manufactures for the isoprene oligomer of OPP.
In addition, Y in formula (Z-1) or (Z-2) is the isoprene oligomer of carboxyl, for example, can by oxidation-type (Z-1) or (Z-2) Y in manufactures for the isoprene oligomer of OPP.
In addition, Y in formula (Z-1) or (Z-2) is the Y in the isoprene oligomer of alkoxycarbonyl, formula (Z-1) or (Z-2) For the isoprene oligomer of alkoxy carbonyl group, for example, can be by the Y in method described above carboxylation formula (Z-1) or (Z-2) The isoprene oligomer of OPP is simultaneously further esterified the isoprene oligomer after the carboxylation and manufactures.
Organic synthesis except the compound represented by formula (Y) and the allyl diphosphonic acid derivative that can select as needed Starting material except, isoprene oligomer of the invention can be manufactured by biosynthesis.Therefore, the present invention accounts for stone The exhaustion of oily resource and environmental problem.
(polyisoprene)
Then, the polyisoprene of the present invention is illustrated.Allyl of the polyisoprene of the present invention represented by formula (X) Compound represented by diphosphonic acid and formula (Y) is synthesized (biosynthesis).It polymerize the allyl diphosphonic acid represented by formula (X) On monomer can be compound and prenyl diphosphate represented by formula (Y) combination.For example, the poly- isoamyl two of the present invention Alkene can by the present invention isoprene oligomer and formula (Y) represented by compound and/or prenyl diphosphate carry out It synthesizes (biosynthesis).For example, the polyisoprene of the present invention can be by unmodified isoprene oligomer and formula (Y) institute The compound of expression is synthesized (biosynthesis), or the chemical combination represented by unmodified isoprene oligomer, formula (Y) Object and prenyl diphosphate are synthesized (biosynthesis).
The polyisoprene of the present invention have the structure close with natural rubber and with rubber molecule highly compatible. The polyisoprene of the present invention also has modified molecular skeleton, therefore it can occur by force with filler (for example, silica) Interaction.Therefore, because the polyisoprene of the present invention and rubber highly compatible, while can be with filler (for example, dioxy SiClx) strong interaction occurs, if the polyisoprene is used for rubber composition, can obtain compared to traditional composition, The rubber composition that its rubber molecule is mixed with filler higher degree.For example, the rubber composition is it is possible thereby to enhance low-heat-generation Property, wet grip performance, wearability, elongation at break and fracture tensile strength.
The skeleton of the polyisoprene of the present invention includes polar group or its analog.Filler is (for example, titanium dioxide as a result, Silicon) dispersion performance when being only present in chain end higher than polar group (for example, end diphosphonic acid group) etc. the case where.Therefore, For example, the improvement increase of low-heat-generation, wet grip performance, wearability, elongation at break and fracture tensile strength.
In addition, the allyl diphosphonic acid represented by the formula (X) is the allyl diphosphonic acid represented by formula (X-1), and formula (X-1) at least one atom or atomic group contained in the parts II in are replaced by other atoms or atomic group, in formula (X-1) In the case that atom or atomic group contained in the parts III are not replaced by other atoms or atomic group, not only bone can be obtained The polyisoprene that frame and chain end are also modified.Because the polyisoprene incessantly there is modified skeleton also to have There is modified end, can preferably interact with filler (for example, silica), if by the polyisoprene For rubber composition, the building rubber compound mixed with filler higher degree compared to traditional composition, rubber molecule can be obtained Object.For example, rubber composition is it is possible thereby to provide the low-heat-generation further enhanced, wet grip performance, wearability, extension at break Rate and fracture tensile strength.
The isoprene list of addition on the allyl diphosphonic acid starting material of formula (X) in the polyisoprene of the present invention Member quantity preferably 30 to 40000, more preferable 31 to 38000, further preferred 1000 to 35000, particularly preferred 2000 to 30000。
For example, the specific example of the polyisoprene of the present invention is poly- represented by formula (ZZ-1) or represented by formula (ZZ-2) At least one atom or atomic group contained in the parts v in isoprene, formula (ZZ-1) or formula (ZZ-2) by other atoms or Atomic group replaces；
Wherein, the n in formula (ZZ-1) indicates integer of 1 to 10；Q indicates 30 to 40000 integer；Y indicate hydroxyl, formoxyl, Carboxyl, alkoxycarbonyl, alkoxy carbonyl or OPP；
Wherein, the n in formula (ZZ-2) indicates integer of 1 to 10；Q indicates 30 to 40000 integer；Y indicate hydroxyl, formoxyl, Carboxyl, alkoxycarbonyl, alkoxy carbonyl or OPP.
Atom or atomic group and other described atoms or atom contained in the parts v in formula (ZZ-1) or formula (ZZ-2) Group can be as described in formula (X-1).
The parts iv in formula (ZZ-1) and (ZZ-2) are the knots for coming from the allyl diphosphonic acid starting material represented by formula (X) Structure.Therefore, the n in formula (ZZ-1) and (ZZ-2) is defined in a manner of identical with the n in formula (X).
Allyl diphosphonic acid starting material of the parts v represented by adding to formula (X) in formula (ZZ-1) and (ZZ-2) Isoprene unit forms.Therefore, the q in formula (ZZ-1) and (ZZ-2) and the allyl diphosphonic acid starting bottom for adding to formula (X) The quantity of the isoprene unit of object is corresponding.
Due to the strong interaction with filler (for example, silica), preferred OPP of Y in formula (ZZ-1) and (ZZ-2), Hydroxyl or carboxyl.
As described above, being found the present invention is based on following：If maintaining the first of the positions 3- in the structure of prenyl diphosphate Structure other than base is constant, even if 3- methyl are replaced by required group, can also be passed through using it naturally occurring Enzyme manufactures isoprene oligomer or polyisoprene.Therefore, the polyisoprene represented by formula (ZZ-1) or formula (ZZ-2) is excellent It is selected as the polyisoprene represented by formula (ZZ-1-1) or represented by formula (ZZ-2-1)：
Wherein, n, q and Y in formula (ZZ-1) are defined in a manner of identical with n, q and Y in formula (ZZ-1), R with formula (Y) same way of the R in defines；
Wherein, n, q and Y in formula (ZZ-2-1) are defined in a manner of identical with n, q and Y in formula (ZZ-2), R with formula (Y) same way of the R in defines.
As described above, it is also preferred that being modified simultaneously to chain end to skeleton incessantly.It is therefore preferable that formula (ZZ-1) or formula (ZZ-2) at least one atom contained by the parts iv in (preferred formula (ZZ-1-1) or formula (ZZ-2-1)) or atomic group are by other Atom or atomic group substitution.
Atom or atom contained in the parts iv in formula (ZZ-1), formula (ZZ-2), formula (ZZ-1-1) or formula (ZZ-2-1) Group and other described atoms or atomic group can be as described in formulas (X-1).
Further, as described above, it is preferred to which the allyl diphosphonic acid represented by formula (X) is the allyl represented by formula (X-1) Base diphosphonic acid, and at least one atom or atomic group contained in the parts II in formula (X-1) are by other atoms or atomic group Replace, atom or atomic group contained in the parts III in formula (X-1) are not replaced by other atoms or atomic group.Therefore, it closes The parts iv in formula (ZZ-1), formula (ZZ-2), formula (ZZ-1-1) and the formula (ZZ-2-1) as represented by following formula (Z), preferred formula (Z) at least one atom contained in the parts iv-II or atomic group are replaced by other atoms or atomic group in, in formula (Z) The parts iv-III contained in atom or atomic group do not replaced by other atoms or atomic group.
In formula (Z) iv-II part or the parts iv-III contained in atom or atomic group and other described atoms or Atomic group can be such as the atom or atomic group as described in formula (X-1).
The concrete example for the parts iv that chain end is also modified may include the structure as represented by above-mentioned formula (a) to (s).Its In, the structure of preferred formula (b), (c), (d), (e), (f), (k), (l) and (r), the structure of more preferable formula (b) and (c), because it There is the stronger interaction with filler (for example, silica), and can more effectively improve low-heat-generation, wet grab Ground performance, wearability, elongation at break and fracture tensile strength.
(method of manufacture polyisoprene)
The polyisoprene of the present invention can be by the change represented by the isoprene oligomer of the present invention and formula (Y) (R-IPP) Object and/or prenyl diphosphate (IPP) are closed, is manufactured by the method (A) of synthesis (biosynthesis) polyisoprene.It can With according to the target physical property of the polyisoprene of acquisition, suitable for change the R-IPP and IPP that use monomer ratio, A kind of percentage of middle monomer can be 0.Alternatively, the polyisoprene of the present invention can be by unmodified isoprene oligomer And the compound represented by formula (Y), it is manufactured by the method (B) of synthesis (biosynthesis) polyisoprene, or by not changing Property isoprene oligomer, compound and prenyl diphosphate represented by formula (Y), it is poly- different by synthesis (biosynthesis) The method (C) of pentadiene manufactures.In addition, in these cases, can according to the target physical property of the polyisoprene of acquisition, Suitable for changing the ratio between R-IPP the and IPP monomers used.
Organic synthesis except the compound represented by formula (Y) and the allyl diphosphonic acid derivative that can select as needed Starting material except, polyisoprene of the invention can be manufactured by biosynthesis.Therefore, the present invention accounts for oil money The exhaustion in source and environmental problem.
All the time, it is known that contain in natural rubber latex (particularly, the natural rubber latex from Para rubber tree) Enzyme and rubber elongation factor (for example, capableing of the enzyme of addition polymerization prenyl diphosphate as described above) or its analog, Activity with the condensation reaction between catalysis isoprene oligomer and prenyl diphosphate, therefore, catalysis successively will be different Pentenyl diphosphonic acid is connected as Z configurations (isoprene unit of new addition is cis-configuration) with isoprene oligomer, generates The reaction of polyisoprene, as follows.
Also, it is known that containing enzyme and rubber elongation factor in the natural rubber latex (rubber latex) from some plant (for example, capableing of the enzyme of addition polymerization prenyl diphosphate as described above) or its analog have catalysis isoprene The activity of condensation reaction between oligomer and prenyl diphosphate, therefore, catalysis successively by prenyl diphosphate with it is different The reaction that pentadiene oligomer is connected as E configuration (isoprene unit of new addition is anti-configuration), generates polyisoprene.
As described above, the methyl when the positions 3- in the structure for replacing prenyl diphosphate with required group and maintenance The structure in addition to 3- methyl of prenyl diphosphate is constant, and obtains the compound of formula (Y) (R-IPP), Ke Yitong It crosses using the compound and using there is the active naturally occurring enzyme for being catalyzed above-mentioned reaction, rubber elongation factor or its is similar Object forms polyisoprene.Therefore, in the present invention, above-mentioned enzyme, rubber elongation factor or its analog can be used to manufacture poly- Isoprene.
In other words, the compound and/or prenyl diphosphate represented by the isoprene oligomer of the present invention and formula (Y) Come synthesize (biosynthesis) present invention polyisoprene method (A), by unmodified isoprene oligomer and formula (Y) Represented compound synthesizes the method (B) of the polyisoprene of (biosynthesis) present invention, by unmodified isoprene Compound and prenyl diphosphate represented by oligomer, formula (Y) synthesize the polyisoprene of (biosynthesis) present invention Method (C), can come by using enzyme, rubber elongation factor or its analog contained in natural rubber latex At.The enzyme cloned from natural rubber latex, rubber elongation factor or its analog can also be used for the above method.
It specifically, can be in the presence of enzyme and/or rubber elongation factor, by the isoamyl two of the present invention in method (A) Alkene oligomer with represented by formula (Y) compound and/or prenyl diphosphate reacted.Similarly, in method (B), It can be in the presence of enzyme and/or rubber elongation factor, by the chemical combination represented by unmodified isoprene oligomer and formula (Y) Object is reacted.Similarly, can be in the presence of enzyme and/or rubber elongation factor in method (C), it will be unmodified different Pentadiene oligomer is reacted with the compound represented by formula (Y), prenyl diphosphate.
More specifically, for example, the reaction can be by represented by the isoprene oligomer containing the present invention and formula (Y) In the solution of compound and/or prenyl diphosphate, adds natural rubber latex or isolated from natural rubber latex Enzyme, rubber elongation factor or its analog and cause.In addition, reaction temperature can be such as 10 to 60 DEG C, the reaction time can be with It it is such as 1 to 72 hour, pH can be such as 6 to 8.In addition it is also possible to as needed, addition magnesium chloride, surfactant, 2- Mercaptoethanol, potassium fluoride and other substances.Even using unmodified isoprene oligomer, reaction can also It is carried out under similarity condition.
In the present invention, the isoprene chain of polyisoprene upper in any direction can extend (anti-configuration or cis- structure Type).In other words, for example, the present invention polyisoprene can be all isoprene units with anti-configuration link gather Isoprene (for example, isoprene oligomer of formula (ZZ-1)), isoprene unit are linked poly- different with trans-cis configuration Pentadiene (for example, polyisoprene of formula (Z-2)) or isoprene unit are linked poly- with Trans-cis-anti-configuration Isoprene.Wherein, the polyisoprene that preferably isoprene unit is linked with trans-cis configuration, because its with it is industrial wide The structure of the natural rubber from Para rubber tree of general application is identical.
In the polyisoprene of the present invention of the reaction manufacture by above description, formula (ZZ-1), formula (ZZ-2), formula (ZZ- 1-1) or the Y in formula (ZZ-2-1) is usually OPP or hydroxyl.OPP comes from IPP or R-IPP.OPP can also be hydrolyzed easily, OPP's Hydrolysis generates hydroxyl.This is why the Y in formula (ZZ-1), (ZZ-2), (ZZ-1-1) or (ZZ-2-1) be usually OPP or Hydroxyl.
In addition, the Y in formula (ZZ-1) or (ZZ-2) is the polyisoprene of formoxyl, for example, can pass through oxidation-type (ZZ-1) Or the Y in (ZZ-2) manufactures for the polyisoprene of OPP.
In addition, Y in formula (ZZ-1) or (ZZ-2) is the polyisoprene of carboxyl, for example, can by oxidation-type (ZZ-1) or (ZZ-2) Y in manufactures for the polyisoprene of OPP.
In addition, Y in formula (ZZ-1) or (ZZ-2) is the Y in the polyisoprene of alkoxycarbonyl, formula (ZZ-1) or (ZZ-2) For the polyisoprene of alkoxy carbonyl group, for example, can be OPP by the Y in method described above carboxylation formula (ZZ-1) or (ZZ-2) Polyisoprene and be further esterified the polyisoprene after the carboxylation and manufacture.
The source of natural rubber latex is not particularly limited.Its example includes：Para rubber tree (Hevea Brasiliensis), rubberplant (Ficus elastica), Ficus lyrata (Ficus lyrata), banyan (Ficus Benjamina), bodhi tree (Ficus religiosa), banyan (Ficus benghalensis), laetarius volemus (Lactarius volemus), sonchus oleraceus (Sonchus oleracers L.), Western dandelion (Taraxacum ) and sunflower (Helianthus annuus) officinale.Wherein, preferred Para rubber tree, because rubber of its manufacture has High molecular weight and latex have high rubber content.
Natural rubber latex, for example, can be by marking channel-shaped with knife or its analog on the trunk of Para rubber tree Wound (this process referred to as " is tapped rubber "), then recycle the natural rubber latex flowed out from cut-off latex dust and obtain.
The enzyme or the rubber elongation factor that are detached from natural rubber latex it can be cited for example that, pass through and centrifuge natural rubber Glue latex and the glue that detaches clear (Serum), bottom phase (bottom fraction) or rubber phase (rubber fraction).Institute It states in clear glue, bottom phase and rubber phase and contains enzyme, rubber elongation factor or its analog.
(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 has outstanding low-heat-generation, wet grip performance, wearability, elongation at break and break-draw strong Degree (especially low-heat-generation and wearability).The polyisoprene of the present invention may be used as rubber components.
In rubber constituent based on 100 mass % meters, the content of polyisoprene of the invention be preferably 20 mass % with On, more preferable 40 mass % or more, further preferred 60 mass % or more.It can be 100 mass %.
Other than the polyisoprene of the present invention, can include as the example for the material that rubber constituent uses：Isoamyl Diene rubber (IR), natural rubber (NR), butadiene rubber (BR), butadiene-styrene rubber (SBR), styrene isoprene butadiene rubber The diene series rubbers such as glue (SIBR), neoprene (CR) and acrylonitrile butadiene rubber (NBR).These rubber materials can be independent It uses, can also be used in combination of two or more.Wherein, preferably NR, BR and SBR.
When by the isoprene oligomer of the present invention in rubber composition, mix for isoprene oligomer The high reason of property, it is preferable to use NR for rubber constituent.By and with the present invention isoprene oligomer and NR, can be more suitably Obtain the effect of the isoprene oligomer of the present invention.
When by the isoprene oligomer of the present invention in rubber composition, it is based in 100 mass % of rubber constituent, NR Content preferably 20 mass % or more, more preferable 40 mass % or more, further preferred 60 mass % or more.It can be 100 matter Measure %.
Relative to 100 mass parts of rubber constituent, it is more than content preferably 1 mass parts of isoprene oligomer of the invention, It is more than more preferable 2 mass parts.When the isoprene oligomer used is less than 1 mass parts, it may not be possible to fully generate its effect.Institute The content for stating isoprene oligomer is preferably also 20 below mass part, and more preferable 15 is below mass part.Higher than 20 mass parts When content, it is likely to reduced intensity and wearability.
The example of the filler used in the present invention includes：Silica, carbon black, clay and calcium carbonate.
The preferred silica of filler used in the present invention.When using silica, can fully it obtain by using this Effect obtained from the isoprene oligomer of invention and/or the polyisoprene of the present invention.Arbitrary silica can be used, Its example includes dry silica (silicic acid anhydride), wet silica (aqueous silicic acid).Preferably wet silica, because It contains more silanol groups.
In the present invention it is also preferred that using carbon black as filler.In this case, it can also fully obtain by using this hair Effect obtained from the polyisoprene of bright isoprene oligomer and/or the present invention.
The manufacture in rubber composition can also suitably be contained other than mentioned component, in rubber composition of the invention In common other compounding agents, for example, silane coupling agent, zinc oxide, stearic acid, various antiaging agents (i.e. antioxidant), soft Agent (for example, oil), wax, vulcanizing agent (for example, sulphur) and vulcanization accelerator.
Rubber composition of the present invention can be prepared by conventional known method.For example, can be by using The rubber mill of open roll, Banbury muller etc. is kneaded the component, then vulcanizes, to manufacture rubber composition.
The rubber composition of the present invention is applicable to tyre element (for example, tyre surface, side wall, tread base, casing ply, slow Rush layer and carcass) with and the like.
(pneumatic tire)
The pneumatic tire of the present invention can be manufactured using the rubber composition according to usual way.It specifically, will not The rubber composition extrusion process of vulcanization is the shape corresponding to tyre element (for example, tyre surface or side wall), then, by usual Method shaped with tyre forming machine, then be bonded with other tyre elements, to form unvulcanized tire, by the unvulcanized wheel Tire carries out hot pressing in vulcanizer, to form tire.
Embodiment
With reference to following embodiment, the present invention is described in detail, but the present invention is not limited only to this.
(manufacture embodiment 1)
(the synthesis (compound (R-IPP) represented by formula (Y)) of 3-R-3- cyclobutenyl diphosphonic acid
The use of n-R- aldehyde is starting material synthesising target compound.By Green et al. (M.B.Green, and W.J.Hickinbotton, J.Chem.Soc.1957,3262) method, in formic acid using dimethylamine n-R- aldehyde alpha-position The outer methylene (compound represented by following formula (i)) of upper introducing.Then, the compound of acquisition is reduced to 2-R- with lithium aluminium hydride reduction Allyl alcohol (compound represented by following formula (ii)).Further, by the compound chlorination it is 2-R- with phosphorus chloride in pyridine Allyl chloride (compound represented by following formula (iii)), then by it in the presence of the Grignard Reagent of fresh synthesis, with titanium dioxide Carbon reaction obtains carboxylic acid (compound represented by following formula (iv)).Then, it restores the compound to form alcohol with lithium aluminium hydride reduction (compound represented by following formula (v)).Then, in pyridine with toluene sulfochloride by the compound carry out tosylation (under Compound represented by formula (vi)).Then, which is subjected to phosphorylation in acetonitrile with trimethyl phosphate, thus to obtain Target product (compound represented by following formula (vii), i.e. compound (R-IPP) represented by formula (Y)).By using TLC and Instrumental Analysis (IR and NMR) confirms the intermediate and final product of each synthesis phase.
In the case where R is respectively ethyl, propyl, butyl, phenyl, sulfydryl, hydroxyl and amino, it is represented formula (Y) has been synthesized Compound (R-IPP).When R is ethyl, propyl, butyl, phenyl, sulfydryl, hydroxyl and amino, the R-IPP of formula (Y) remembers respectively It is R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G.
(embodiment 1)
(manufacture (alltrans) of isoprene oligomer)
The isoprene oligomer that whole isoprene units are linked with anti-configuration is prepared, as represented by formula (Z-1-1-1).
(preparation of transformant)
First, transformant is prepared.In the preparation of transformant, importing mankind's geranyl geranyl diphosphate synzyme is used pET15b(pET15b/human-GGPS).The pET15b/human-GGPS by multi-component Science Institute of Northeastern University assistant Upper rich associate professor (Associate Professor Hiroshi SAGAMI, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University) it provides.
Pass through heat shock Transformed E .coli BL21 (DE3) using pET15b/human-GGPS.The transformant is coated on and is contained After having on the LB agar mediums of the ampicillin of 50 μ g/mL, then overnight incubation at 37 DEG C selects transformant.
(manufacture with the active protein of prenyltransferases)
Obtained E.coli BL21 (DE3)/pET15b/human-GGPS (wild type) is inoculated into and contains 50 μ g/ equipped with 3mL In the test tube of the LB culture mediums of the ampicillin of mL, shaken cultivation 5 hours at 37 DEG C.In obtained culture solution, 1mL etc. is taken Divide and is seeded in the 500mL conical flasks of the LB culture mediums containing 50 μ g/mL ampicillins equipped with 100mL, it will at 37 DEG C Cell oscillation culture 3 hours.Then, addition IPTG is to its a concentration of 0.1mmol/L, then by cell oscillation culture at 30 DEG C 18 hours.The culture solution is centrifuged, wet thallus is obtained.After being homogenized the wet thallus by ultrasonic wave, centrifuges and obtain supernatant Liquid.Being purified from the supernatant using HisTrap (Amersham companies) has the active protein of prenyltransferases. The SDS-PAGE that is purified by of protein is confirmed.
(preparation of isoprene oligomer)
It prepares containing protein 10mg, 50mM Tris-HCl buffer solutions (pH 7.5), 40mM magnesium chlorides, 25mM 2- after purification Mercaptoethanol, 1mM starting materials (geranyl diphosphonic acid (GPP) and 1mM prenyl diphosphates (IPP) or manufacture embodiment A kind of reaction solution in 1 in manufactured R-IPP.The reaction 1 hour is carried out in 37 DEG C of water-bath.After reaction, Saturated salt solution 100ml and pentane 500ml is added to be stood after stirring the mixture.Then, by being concentrated by evaporation supernatant Liquid (pentane layer) is extremely dry.It takes remnant parts to confirm product structure by NMR, is isoprene oligomer.Thus it obtains Isoprene oligomer concrete condition (n, m and R in formula (Z-1-1-1)) it is as shown in table 1.Herein, it is risen based on what is used The information of beginning substrate and the isoprene chain length measured with TLC, calculate the n and m in formula (Z-1-1-1).In addition, formula (Z-1-1- 1) the R structures in are identified by NMR, TLC and GC-MS.
In addition, using monomer IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R- respectively The isoprene oligomer that IPP-G is obtained, is referred to as isoprene oligomer (t- controls), isoamyl in aftermentioned experiment use Diene 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).In addition, isoamyl in experiment Diene oligomer (t-E), isoprene oligomer (t-F) add respectively as isoprene oligomer a and isoprene oligomer e Enter in rubber composition.
[table 1]
(relative activity of monomer)
The one kind for making IPP or manufacturing in the R-IPP in embodiment 1 carries out instead under the following conditions with geranyl diphosphonic acid (GPP) It answers.Each R-IPP indicates the relative activity of GPP with index relative to IPP active (=100).
It prepares containing protein 500ng, 50mM Tris-HCl buffer solutions (pH 7.5), 40mM magnesium chlorides, 25mM after purification 2 mercapto ethanol, 12.5 μM GPP and 50 μM of [1-14C] one kind in IPP or manufacture embodiment 1 in the R-IPP for preparing. The reaction 1 hour is carried out in 37 DEG C of water-bath.After reaction, is counted using liquid scintillation and measure work in each condition with TLC Property.Relative to the activity (=100) of IPP, the relative activity of each R-IPP is as shown in table 2.
Isoprene oligomer is pure using being carried out with the same mode in 1 part of embodiment (preparation of isoprene oligomer) Change.Then, the concrete condition of the isoprene oligomer of acquisition (n, m and R in formula (Z-1-1-1)) is shown in table 2.This Place, information based on the starting material used and the isoprene chain length measured with TLC, n and m in calculating formula (Z-1-1-1). In addition, the R structures in formula (Z-1-1-1) are identified by NMR, TLC and GC-MS.
[table 2]
Table 1 and 2 the results show that even if using R-IPP acquisitions isoprene oligomer if having with when using IPP Identical molecular weight.Be modified corresponding with the R-IPP used has occurred in the skeleton for also demonstrating acquisition isoprene oligomer.
(embodiment 2)
(preparation of polyisoprene)
Use the isoprene oligomer linked with anti-configuration such as whole isoprene units represented by formula (Z-1-1-1) (all trans-) prepares polyisoprene.
It prepares containing 10 μ L latex components, 50mM Tris-HCl buffer solutions (pH 7.5), 25mM magnesium chlorides, 40mM2- sulfydryl second One kind in alcohol, 40mM potassium fluorides, 50 μM of isoprene oligomer and 1mM IPP or manufacture embodiment 1 in the R-IPP for preparing Reaction solution.The reaction 3 days is carried out in 30 DEG C of water-bath.After reaction, molecular weight is measured by GPC.Then, it is based on molecule The information of the numerical value of amount and the starting material used calculates the quantity for adding to the isoprene unit on starting material GPP.Knot Fruit is as shown in table 3.
Latex component used herein is by clear to obtaining the glue obtained by latex carries out ultracentrifugation from Para rubber tree.
The isoprene oligomer used is isoamyl two prepared in above-mentioned part (preparation of isoprene oligomer) Alkene oligomer (t- controls), 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) one kind in.
[table 3]
Table 3 the results show that even if using R-IPP acquisitions polyisoprene if with having the same point when using IPP Son amount.Further, the polyisoprene of acquisition is analyzed with NMR, TLC and GC-MS, it is thus identified that oligomeric with isoprene The case where object, is similar, and the skeleton of polyisoprene has carried out be modified corresponding with used R-IPP.
(embodiment 3)
(manufacture (Trans-cis) of isoprene oligomer)
The isoprene oligomer that isoprene unit is linked with trans-cis configuration is prepared, as represented by formula (Z-2-1-1).
(preparation of transformant)
First, transformant is prepared.In the preparation of transformant, 11 isopentenyl diphosphates for importing micrococcus luteus B-P26 are used The pET22b (pET22b/MLU-UPS) of synthase is used as dsDNA templates.The pET22b/MLU-UPS is by Northeastern University's multi-component The ancient mountain kind person of outstanding talent of Science Institute teaches (Prof.Tanetoshi Koyama, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University) it provides.
Pass through heat shock Transformed E .coli BL21 (DE3) using pET22b/MLU-UPS.The transformant is coated on containing 50 After on the LB agar mediums of the ampicillin of μ g/mL, then overnight incubation at 37 DEG C selects transformant.
(manufacture with the active protein of prenyltransferases)
Obtained E.coli BL21 (DE3)/pET22b/MLU-UPS is inoculated into the ammonia benzyl mould for containing 50 μ g/mL equipped with 3mL In the test tube of the LB culture mediums of element, shaken cultivation 5 hours at 37 DEG C.In obtained culture solution, takes 1mL deciles to be seeded to and be equipped with In the 500mL conical flasks of the LB culture mediums containing 50 μ g/mL ampicillins of 100mL, by cell oscillation culture 3 at 37 DEG C Hour.Then, addition IPTG is to its a concentration of 0.1mmol/L, then by cell oscillation culture 18 hours at 30 DEG C.Centrifugation point From the culture solution, wet thallus is obtained.After being homogenized the wet thallus by ultrasonic wave, centrifuges and obtain supernatant.Use HisTrap (Amersham companies) purifying from the supernatant has the active protein of prenyltransferases.The purifying of protein is logical SDS-PAGE is crossed to be confirmed.
(preparation of isoprene oligomer)
It prepares containing protein 10mg, 50mM Tris-HCl buffer solutions (pH 7.5), 40mM magnesium chlorides, 40mM after purification (farnesyl diphosphonic acid (FPP) and 1mM IPP or manufacture are real for Triton X-100,25mM 2 mercapto ethanols, 1mM starting materials Apply one kind in the R-IPP prepared in example 1.The reaction 1 hour is carried out in 37 DEG C of water-bath.After reaction, saturation is added Saline solution 100ml and n-butyl alcohol 500ml are stood after stirring the mixture.Then, by being concentrated by evaporation supernatant (1- fourths Alcohol layer) it is extremely dry.It takes remnant parts to confirm product structure by NMR, is isoprene oligomer.Obtained isoprene The concrete condition (n, m and R in formula (Z-2-1-1)) of oligomer is as shown in table 4.Herein, the letter based on the starting material used Breath and the isoprene chain length measured with TLC, calculate the n and m in formula (Z-2-1-1).In addition, the R structures in formula (Z-2-1-1) It is identified by NMR, TLC and GC.
In addition, using monomer IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R- respectively IPP-G and the isoprene oligomer obtained, be referred to as in aftermentioned experiment use isoprene oligomer (tc- controls), 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).In addition, In experiment, isoprene oligomer (tc-E) is added as isoprene oligomer (b) in rubber composition.In addition, in experiment, Each isoprene oligomer (tc-A), isoprene oligomer (tc-B), isoprene oligomer (tc-C), isoprene are low Polymers (tc-D), isoprene oligomer (tc-E), isoprene oligomer (tc-F) and isoprene oligomer (tc-G) point It Zuo Wei not isoprene oligomer h, isoprene oligomer i, isoprene oligomer j, isoprene oligomer k, isoamyl two Alkene oligomer l, isoprene oligomer m and isoprene oligomer n are added in rubber composition.
[table 4]
(relative activity of monomer)
The one kind for making IPP or manufacturing in the R-IPP in embodiment 1 carries out instead under the following conditions with farnesyl diphosphonic acid (FPP) It answers.Each R-IPP is indicated the relative activity of FPP with the index relative to IPP active (=100).
It prepares containing protein 500ng, 50mM Tris-HCl buffer solutions (pH 7.5), 40mM magnesium chlorides, 40mM after purification Triton X-100,25mM 2 mercapto ethanols, 12.5 μM FPP and 50 μM of [1-14C] IPP or manufacture embodiment 1 in prepare R-IPP in a kind of reaction solution.The reaction 1 hour is carried out in 37 DEG C of water-bath.After reaction, using liquid scintillation meter It counts and measures activity in each condition with TLC.Relative to the activity (=100) of IPP, relative activity such as 5 institute of table of each R-IPP Show.
Isoprene oligomer is pure using being carried out with the same mode in 3 part of embodiment (preparation of isoprene oligomer) Change.Then, the concrete condition of the isoprene oligomer of acquisition (n, m and R in formula (Z-2-1-1)) is shown in table 5.This Place, information based on the starting material used and the isoprene chain length measured with TLC, n and m in calculating formula (Z-2-1-1). In addition, the R structures in formula (Z-2-1-1) are identified by NMR, TLC and GC-MS.
[table 5]
Table 4 and 5 the results show that even if using R-IPP acquisitions isoprene oligomer if having with when using IPP Identical molecular weight.Be modified corresponding with the R-IPP used has occurred in the skeleton for also demonstrating acquisition isoprene oligomer.
(embodiment 4)
(preparation of polyisoprene)
Use the isoprene oligomer linked with trans-cis configuration such as the isoprene unit represented by formula (Z-2-1-1) (Trans-cis) prepares polyisoprene.
It prepares containing 10 μ L latex components, 50mM Tris-HCl buffer solutions (pH 7.5), 25mM magnesium chlorides, 40mM2- sulfydryl second One kind in alcohol, 40mM potassium fluorides, 50 μM of isoprene oligomer and 1mM IPP or manufacture embodiment 1 in the R-IPP for preparing Reaction solution.The reaction 3 days is carried out in 30 DEG C of water-bath.After reaction, molecular weight is measured by GPC.Then, it is based on molecule The information of the numerical value of amount and the starting material used calculates the quantity for adding to the isoprene unit on starting material FPP.Knot Fruit is shown in table 6.
Latex component used herein is by clear to obtaining the glue obtained by latex carries out ultracentrifugation from Para rubber tree.
The isoprene oligomer used is isoamyl two prepared in above-mentioned part (preparation of isoprene oligomer) Alkene oligomer (tc- controls), 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 isoamyl two One kind in alkene oligomer (tc-G).In addition, in subsequent experiment, isoprene oligomer (tc-E), isoamyl two are used Rubber composition is added respectively as polyisoprene D and polyisoprene A in the polyisoprene of alkene oligomer (tc-F) manufacture In.In addition, each use isoprene oligomer (tc-A), isoprene oligomer (tc-B), isoprene oligomer (tc- C), isoprene oligomer (tc-D), isoprene oligomer (tc-E), isoprene oligomer (tc-F), isoprene are low The polyisoprene of polymers (tc-G) manufacture is respectively as polyisoprene I, polyisoprene J, polyisoprene K, poly- isoamyl Diene L, polyisoprene M, polyisoprene N, polyisoprene P are added in rubber composition.
[table 6]
Table 6 the results show that even if using R-IPP acquisitions polyisoprene if with having the same point when using IPP Son amount.Further, the polyisoprene of acquisition is analyzed with NMR, TLC and GC-MS, it is thus identified that oligomeric with isoprene The case where object, is similar, and the skeleton of polyisoprene has carried out be modified corresponding with used R-IPP.
(embodiment 5)
(manufacture (all trans-) of isoprene oligomer)
The isoprene oligomer that whole isoprene units are linked with anti-configuration is prepared, as represented by formula (Z-1-1-1).
(preparation of transformant)
First, transformant is prepared.In the preparation of transformant, importing bacillus stearothermophilus farnesyl diphosphate synthase is used PET15b (pET15b/bacillus-FPS).The pET15b/bacillus-FPS is by Northeastern University's multi-component scientific research Ancient mountain kind person of outstanding talent teach (Prof.Tanetoshi Koyama, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University) it provides.
Pass through heat shock Transformed E .coli BL21 (DE3) using pET15b/bacillus-FPS.The transformant is coated on After on the LB agar mediums of ampicillin containing 50 μ g/mL, then overnight incubation at 37 DEG C selects transformant.
(manufacture with the active protein of prenyltransferases)
Obtained E.coli BL21 (DE3)/pET15b/bacillus-FPS (wild type) is inoculated into and contains 50 μ equipped with 3mL In the test tube of the LB culture mediums of the ampicillin of g/mL, shaken cultivation 5 hours at 37 DEG C.In obtained culture solution, 1mL is taken Decile is seeded in the 500mL conical flasks of the LB culture mediums containing 50 μ g/mL ampicillins equipped with 100mL, at 37 DEG C By cell oscillation culture 3 hours.Then, addition IPTG then trains cell oscillation at 30 DEG C to its a concentration of 0.1mmol/L It supports 18 hours.The culture solution is centrifuged, wet thallus is obtained.After being homogenized the wet thallus by ultrasonic wave, centrifuge on obtaining Clear liquid.Being purified from the supernatant using HisTrap (Amersham companies) has the active albumen of prenyltransferases Matter.The SDS-PAGE that is purified by of protein is confirmed.
(preparation of isoprene oligomer)
It prepares containing protein 10mg, 50mM Tris-HCl buffer solutions (pH 7.5), 40mM magnesium chlorides, 25mM 2- after purification Mercaptoethanol, 1mM starting materials (dimethylallyl diphosphate (DMAPP) and 1mM prenyl diphosphates (IPP) or system Make a kind of reaction solution in the R-IPP prepared in embodiment 1.The reaction 1 hour is carried out in 37 DEG C of water-bath.Reaction knot Shu Hou is added saturated salt solution 100ml and pentane 500ml and is stood after stirring the mixture.Then, pass through evaporation and concentration Supernatant (pentane layer) is extremely dry.It takes remnant parts to confirm product structure by NMR, is isoprene oligomer.It obtains Isoprene oligomer concrete condition (n, m and R in formula (Z-1-1-1)) it is as shown in table 7.Herein, it is risen based on what is used The information of beginning substrate and the isoprene chain length measured with TLC, calculate the n and m in formula (Z-1-1-1).In addition, formula (Z-1-1- 1) the R structures in are identified by NMR, TLC and GC-MS.
In addition, using monomer IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R- respectively IPP-G and the isoprene oligomer obtained, be referred to as in aftermentioned experiment use isoprene oligomer (t- controls -1), Isoprene oligomer (t-A-1), isoprene oligomer (t-B-1), isoprene oligomer (t-C-1), isoprene are low Polymers (t-D-1), isoprene oligomer (t-E-1), isoprene oligomer (t-F-1) and isoprene oligomer (t-G- 1)。
[table 7]
(relative activity of monomer)
Make IPP or manufactures one kind in embodiment 1 in the R-IPP for preparing with dimethyl diphosphonic acid (DMAPP) under the following conditions It is reacted.Each R-IPP indicates the relative activity of DMAPP with index relative to IPP active (=100).
It prepares containing protein 500ng, 50mM Tris-HCl buffer solutions (pH 7.5), 40mM magnesium chlorides, 25mM after purification 2 mercapto ethanol, 12.5 μM DMAPP and 50 μM of [1-14C] one kind in IPP or manufacture embodiment 1 in the R-IPP for preparing Reaction solution.The reaction 1 hour is carried out in 37 DEG C of water-bath.After reaction, is counted using liquid scintillation and measured with TLC Activity under the conditions of each.Relative to the activity (=100) of IPP, the relative activity of each R-IPP is as shown in table 8.
Isoprene oligomer is pure using being carried out with the same mode in 5 part of embodiment (preparation of isoprene oligomer) Change.Then, the concrete condition of the isoprene oligomer of acquisition (n, m and R in formula (Z-1-1-1)) is shown in table 8.This Place, information based on the starting material used and the isoprene chain length measured with TLC, n and m in calculating formula (Z-1-1-1). In addition, the R structures in formula (Z-1-1-1) are identified by NMR, TLC and GC-MS.
[table 8]
Table 7 and 8 the results show that even if using R-IPP acquisitions isoprene oligomer if having with when using IPP Identical molecular weight.Be modified corresponding with the R-IPP used has occurred in the skeleton for also demonstrating acquisition isoprene oligomer.
(embodiment 6)
(preparation of polyisoprene)
Then, the isoprene linked with anti-configuration using whole isoprene units as represented by formula (Z-1-1-1) is low Polymers (all trans-) prepares polyisoprene.
It prepares containing 10 μ L latex components, 50mM Tris-HCl buffer solutions (pH 7.5), 25mM magnesium chlorides, 40mM2- sulfydryl second One kind in alcohol, 40mM potassium fluorides, 50 μM of isoprene oligomer and 1mM IPP or manufacture embodiment 1 in the R-IPP for preparing Reaction solution.The reaction 3 days is carried out in 30 DEG C of water-bath.After reaction, molecular weight is measured by GPC.Then, it is based on molecule The information of the numerical value of amount and the starting material used calculates the quantity for adding to the isoprene unit on starting material DMAPP. The results are shown in Table 9.
Latex component used herein is by clear to obtaining the glue obtained by latex carries out ultracentrifugation from Para rubber tree.
The isoprene oligomer used is isoamyl two prepared in above-mentioned part (preparation of isoprene oligomer) Alkene oligomer (t- controls -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 different One kind in pentadiene oligomer (t-G-1).
[table 9]
Table 9 the results show that even if using R-IPP acquisitions polyisoprene if with having the same point when using IPP Son amount.Further, the polyisoprene of acquisition is analyzed with NMR, TLC and GC-MS, it is thus identified that oligomeric with isoprene The case where object, is similar, and the skeleton of polyisoprene has carried out be modified corresponding with used R-IPP.
(embodiment 7)
(manufacture (Trans-cis) of isoprene oligomer)
Then, using allyl diphosphonic acid derivative, prepare such as isoprene unit represented by formula (Z-2-1-1) with it is trans-- Cis-configuration connection and not only skeleton but also chain end also occur modified isoprene oligomer (skeleton and chain end change The isoprene oligomer of property).
(preparation of allyl diphosphonic acid derivative)
First, synthesis of allyl diphosphonic acid derivative.
(manufacture embodiment 2)
(the conjunction of-ten two carbon of 8- methoxyl group -3,7- dimethyl-(2E, 6E)-dialkylene diphosphonic acid (compound represented by formula (B)) At)
The use of geraniol is starting material synthesising target compound.With pyridine and acetic anhydride to geraniol in anhydrous methylene chloride Acetylation is carried out, acetic acid esters (compound represented by following formula (bi)) (yield 95%) is obtained.Then, by the positions the 8- of the acetic acid esters Carbon carry out selenolite in ethanol, obtain aldehyde body (compound represented by following formula (bii)) (yield 24%).Then, hydrogen is used The aldehyde is carried out basic hydrolysis by potassium oxide, forms alcohol (compound represented by following formula (biii)) (yield 38%).Then, exist The alcohol is handled with imidazoles, tert-butyl diphenyl chlorosilane (TBDPS) in anhydrous methylene chloride, obtains changing shown in following (biv) Close object (yield 80%).Then, so that the compound is reacted with butyl lithium in absolute ether, obtain butyl alcohol (following formula (bv) institute The compound of expression) (yield 73%).Then, in anhydrous tetrahydro furan, the compound is converted to sodium salt, therewith in the sodium Methyl iodide is added in salt and by Williamson synthesis, obtains ether body (compound represented by following (bvi)) (yield 95%). Then, the ether is subjected to elimination reaction using four positive ammonium fluorides in anhydrous tetrahydro furan, obtains alcohol (following formula (bvii) institute table The compound shown) (yield 87%).Then, N- chloro bromosuccinimides are used in -40 DEG C or less, anhydrous methylene chloride solvent Chlorine substitution is carried out to primary hydroxyl with dimethyl sulphide, obtains chloride (compound represented by following (bviii)) (yield 92%). Then, diphosphate is carried out to the chloride with three (tetra-n-butyl ammoniums) hydrogenation diphosphonic acid in anhydrous acetonitrile, obtains target production Object (compound represented by following formula (bix), i.e. compound represented by formula (B)) (yield 26%).
The confirmation of the intermediate and final product of each synthesis phase is carried out using TLC and Instrumental Analysis (IR and NMR).
(manufacture embodiment 3)
(the conjunction of-ten two carbon of 8- hydroxyl -3,7- dimethyl-(2E, 6E)-dialkylene diphosphonic acid (compound represented by formula (C)) At)
The use of geraniol is starting material synthesising target compound.With pyridine and acetic anhydride to geraniol in anhydrous methylene chloride Acetylation is carried out, acetic acid esters (compound represented by following formula (ci)) (yield 97%) is obtained.Then, by the positions the 8- of the acetic acid esters Carbon carry out selenolite in ethanol, obtain aldehyde body (compound represented by following formula (cii)) (yield 20%).Then, hydrogen is used The aldehyde is carried out basic hydrolysis by potassium oxide, forms alcohol (compound represented by following formula (ciii)) (yield 42%).Then, exist The alcohol is handled with imidazoles, tert-butyl diphenyl chlorosilane (TBDPS) in anhydrous methylene chloride, obtains changing shown in following (civ) Close object (yield 80%).Then, so that the compound is reacted with butyl lithium in anhydrous ether, obtain butanol body (yield 62%). Then, it uses four positive ammonium fluorides by the pure carry out elimination reaction in anhydrous tetrahydro furan, obtains two alcohols (following formula (cvi) institute The compound of expression) (yield 94%).Then, sub- with N- chloro succinic acids acyl in -40 DEG C or less, anhydrous methylene chloride solvent Amine carries out chlorine substitution with dimethyl sulphide to primary hydroxyl, obtains chloride (compound represented by following (cvii)) (yield 90%).Then, diphosphate is carried out to the chloride with three (tetra-n-butyl ammoniums) hydrogenation diphosphonic acid in anhydrous acetonitrile, obtained Target product (compound represented by following formula (cviii), i.e. compound represented by formula (C)) (yield 46%).
The confirmation of the intermediate and final product of each synthesis phase is carried out using TLC and Instrumental Analysis (IR and NMR).
(preparation of mutant enzyme)
Using allyl diphosphonic acid derivative as starting material, even if there are prenyltransferases using wild type In the case of active enzyme, reaction can also be carried out and prepare isoprene oligomer.However, in order to improve reaction efficiency, prepare Improve the variant enzyme to the enzymatic activity of allyl diphosphonic acid derivative.
The reagent used comes from QuickChange site-directed mutagenesis kits (QuickChange Site-Directed Mutagenesis Kit) (Stratagene companies).Design primer is mutated so as to can be introduced in target site.Mutation is drawn Object is purchased from company of Institute of Health on Nutriology (MEDICAL＆BIOLOGICAL LABORATORIES CO., LTD) (manufacturer： IDT).The primer of design is as follows.
Prepare the primer of 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)
Prepare the primer of 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)
DsDNA templates using import from micrococcus luteus B-P26 11 prenyl diphosphate synthases (with Under, also referred to as wild-type enzyme) pET22b (pET22b/MLU-UPS).The pET22b/MLU-UPS is by Northeastern University's multi-component The ancient mountain kind person of outstanding talent of Science Institute teaches (Prof.Tanetoshi Koyama, Institute of Multidisciplinary Research for Advanced Materials, Tohoku University) it provides.Mix 10x Pfu polymerases buffering 2 μ L of liquid, dsDNA templates 2-20ng, forward primer 50ng, 0.4 μ L of reverse primer 50ng, dNTPs (each is 2.5mM), ddH2O totally 20 μ L, Pfu polymerase (2.5U/ μ l) 0.4mL carry out PCR reactions.The condition of PCR reactions is as follows：
95 DEG C, 30 seconds, a cycle；
95 DEG C, 30 seconds/55 DEG C, 1 minute/68 DEG C, 8 minutes, 15 cycles.
After PCR, 0.4 μ L Dpn I are added in PCR solution, carry out at 37 DEG C 1 hour place to the PCR solution with Dpn I Reason.Using the Dpn I treatment fluids of 1 to 10 μ L, pass through heat shock Transformed E .coli DH5 α.By the transformant be coated on containing After on the LB agar mediums of the ampicillin of 50 μ g/mL, then overnight incubation at 37 DEG C selects transformant.Then, exist The transformant is cultivated in the LB culture mediums of ampicillin containing 50 μ g/mL to stay overnight, and passes through alkali SDS from obtained culture solution Legal system prepared plasmid.The mutation of the plasmid is confirmed using sequenator.
(manufacture with the active protein of prenyltransferases)
Obtained E.coli BL21 (DE3)/pET22b/MLU-UPS (wild and saltant type) is inoculated into and contains 50 μ equipped with 3mL In the test tube of the LB culture mediums of the ampicillin of g/mL, shaken cultivation 5 hours at 37 DEG C.In obtained culture solution, 1mL is taken Decile is seeded in the 500mL conical flasks of the LB culture mediums containing 50 μ g/mL ampicillins equipped with 100mL, at 37 DEG C The shaken cultivation cell 3 hours.Then, addition IPTG is to its a concentration of 0.1mmol/L, and then this is thin for shaken cultivation at 30 DEG C Born of the same parents 18 hours.The culture solution is centrifuged, wet thallus is obtained.After being homogenized the wet thallus by ultrasonic wave, centrifuge on obtaining Clear liquid.Being purified from the supernatant using HisTrap (Amersham companies) has the active albumen of prenyltransferases Matter.The SDS-PAGE that is purified by of protein is confirmed.The amino acid of thus obtained variant enzyme N77A and variant enzyme L91D The SEQ ID NO of sequence such as sequence table：Shown in 5 and 6.
(preparation of isoprene oligomer)
It prepares containing protein 10mg, 50mM Tris-HCl buffer solutions (pH 7.5), 40mM magnesium chlorides, 40mM after purification Triton X-100,25mM 2 mercapto ethanols, 1mM starting materials are (in farnesyl diphosphonic acid (FPP) or manufacture embodiment 2 and 3 One in the R-IPP prepared in one kind and 1mM IPP or manufacture embodiment 1 in the allyl diphosphonic acid derivative of preparation The reaction solution of kind.The reaction 1 hour is carried out in 37 DEG C of water-bath.After reaction, saturated salt solution 100ml and 1- is added Butanol 500ml is stood after stirring the mixture.Then, by being concentrated by evaporation supernatant (n-butyl alcohol layer) to drying.It takes Remnant parts confirm product structure by NMR, are isoprene oligomer.The specific feelings of obtained isoprene oligomer Condition (n, m and R in formula (Z-2-1-1)) is as shown in table 10.Herein, it information based on the starting material used and is measured with TLC Isoprene chain length, calculate the n and m in formula (Z-2-1-1).In addition, R structures in formula (Z-2-1-1) by NMR, TLC and GC-MS is identified.
In addition, using variant enzyme N77A as enzyme, using the compound represented by formula (B) as starting material, monomer is used respectively The isoprene that IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G are obtained is low Polymers is referred to as isoprene oligomer (tc- controls-AB), isoprene oligomer (tc-A- in aftermentioned experiment use 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).
In addition, using variant enzyme L91D as enzyme, using the compound represented by formula (B) as starting material, monomer is used respectively The isoprene that IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G are obtained is low Polymers is referred to as isoprene oligomer (tc- controls-DB), isoprene oligomer (tc-A- in aftermentioned experiment use 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).
In addition, using variant enzyme N77A as enzyme, using the compound represented by formula (C) as starting material, monomer is used respectively The isoprene that IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G are obtained is low Polymers is referred to as isoprene oligomer (tc- controls-AC), isoprene oligomer (tc-A- in aftermentioned experiment use 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).
In addition, using variant enzyme L91D as enzyme, using the compound represented by formula (C) as starting material, monomer is used respectively The isoprene that IPP, R-IPP-A, R-IPP-B, R-IPP-C, R-IPP-D, R-IPP-E, R-IPP-F and R-IPP-G are obtained is low Polymers is referred to as isoprene oligomer (tc- controls-DC), isoprene oligomer (tc-A- in aftermentioned experiment use 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).
In addition, in experiment, each isoprene oligomer (tc-E-AB), isoprene oligomer (tc-F-AB), isoprene Oligomer (tc-E-DB) and isoprene oligomer (tc-F-DB) are oligomeric respectively as isoprene oligomer c, isoprene Object f, isoprene oligomer d, isoprene oligomer g are added in rubber composition.
[table 10]
(relative activity of monomer)
Make IPP or manufacture one kind in the R-IPP prepared in embodiment 1, with farnesyl diphosphonic acid (FPP) or manufacture embodiment 2 With 3 in one kind in the allyl diphosphonic acid derivative for preparing reacted under the following conditions.It is respectively relative to FPP and IPP Activity, by the relative activity of each allyl diphosphonic acid derivative and each R-IPP with exponential representation, wherein FPP and IPP Activity be set to 100.
It prepares containing protein 500ng, 50mM Tris-HCl buffer solutions (pH7.5), 40mM magnesium chlorides, 40mM after purification The allyl diphosphonic acid prepared in Triton X-100,25mM 2 mercapto ethanols, 12.5 μM of FPP or manufacture embodiment 2 and 3 spreads out One kind in biology and 50 μM of [1-14C] a kind of reaction solution in IPP or manufacture embodiment 1 in the R-IPP for preparing. The reaction 1 hour is carried out in 37 DEG C of water-bath.After reaction, is counted using liquid scintillation and measure work in each condition with TLC Property.Relative to the activity (=100) of IPP, the relative activity of allyl diphosphonic acid derivative and R-IPP are as shown in table 11.
Isoprene oligomer uses pure with the same manner progress in 7 part of embodiment (preparation of isoprene oligomer) Change.Then, the concrete condition of the isoprene oligomer of acquisition (n, m and R in formula (Z-2-1-1)) is shown in table 11.This Place, information based on the starting material used and the isoprene chain length measured with TLC, n and m in calculating formula (Z-2-1-1). In addition, the R structures in formula (Z-2-1-1) are identified by NMR, TLC and GC-MS.
[table 11]
Table 10 and 11 the results show that even if using R-IPP and allyl diphosphonic acid derivative, the isoprene of acquisition is low Polymers also with molecular weight having the same when using IPP and FPP.Acquisition isoprene oligomer is also demonstrated to have occurred and use R-IPP and the corresponding modification of allyl diphosphonic acid derivative.
(embodiment 8)
(preparation of polyisoprene)
Then, polyisoprene is prepared using following isoprene oligomer：It is prepared using allyl diphosphonic acid derivative, such as formula (Z-2-1-1) represented by, isoprene unit is linked with trans-cis configuration and not only skeleton but also chain end is also sent out Raw modified isoprene oligomer (the isoprene oligomer that skeleton and chain end are modified).
It prepares containing 10 μ L latex components, 50mM Tris-HCl buffer solutions (pH 7.5), 25mM magnesium chlorides, 40mM 2- sulfydryl second In alcohol, 40mM potassium fluorides, 50 μM of isoprene oligomer and 1mM IPP or manufacture embodiment 1 in the R-IPP for preparing one The reaction solution of kind.The reaction 3 days is carried out in 30 DEG C of water-bath.After reaction, molecular weight is measured by GPC.Then, it is based on dividing The information of the numerical value and the starting material used of son amount, calculating add on starting material FPP or allyl diphosphonic acid derivative Isoprene unit quantity.As a result as shown in table 12.
Latex component used herein is by clear to obtaining the glue obtained by latex carries out ultracentrifugation from Para rubber tree.
The isoprene oligomer used is isoamyl two prepared in above-mentioned part (preparation of isoprene oligomer) Alkene oligomer (tc- controls-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 are oligomeric Object (tc-F-AB), isoprene oligomer (tc-G-AB), isoprene oligomer (tc- controls-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), isoprene oligomer (tc-G- DB), isoprene oligomer (tc- controls-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), It is isoprene oligomer (tc-F-AC), isoprene oligomer (tc-G-AC), isoprene oligomer (tc- controls-DC), different Pentadiene oligomer (tc-A-DC), isoprene oligomer (tc-B-DC), isoprene oligomer (tc-C-DC), isoamyl two Alkene oligomer (tc-D-DC), isoprene oligomer (tc-E-DC), isoprene oligomer (tc-F-DC) and isoprene are low One kind in polymers (tc-G-DC).
It is each to use isoprene oligomer (tc-E-AB), isoprene oligomer (tc-F-AB), isoamyl two in aftermentioned experiment Alkene oligomer (tc-E-DB), isoprene oligomer (tc-F-DB), isoprene oligomer (tc-E-AC) and isoprene are low The polyisoprene of polymers (tc-E-DC) manufacture is respectively as polyisoprene E, polyisoprene B, polyisoprene F, poly- different Pentadiene C, polyisoprene G, polyisoprene H are added in rubber composition.
[table 12]
Table 12 the results show that even if the isoprene oligomer being modified using R-IPP and skeleton and chain end, acquisition Polyisoprene also with molecular weight having the same when using IPP and unmodified isoprene oligomer.Further, to obtaining Polyisoprene analyzed with NMR, TLC and GC-MS, it is thus identified that with isoprene oligomer the case where is similar, gathers different Pentadiene has carried out modification corresponding with the isoprene oligomer of used R-IPP and the modification of skeleton and chain end.
The chain end of the isoprene oligomer and polyisoprene that are prepared in embodiment is (with formula (Z-1), formula (Z-2), formula (ZZ-1) or the Y of formula (ZZ-2) is corresponding) it is hydroxyl or OPP.
Chemical reagent for embodiment 9 to 41 and comparative example 1 to 4 is listed below.
NR：TSR20
BR：BR01 (JSR companies)
Carbon black：DIABLACK (N220) (Mitsubishi Chemical Ind)
Isoprene oligomer a to n：Prepared isoprene oligomer in above-described embodiment
Polyisoprene A to P：Prepared polyisoprene in above-described embodiment
Zinc oxide：Zinc oxide #1 (mining company of Mitsui Metal Co., Ltd.)
Stearic acid：Stearic acid (You companies)
Antiaging agent：(the emerging chemical industry of imperial palace is public by NOCRAC 6C (N- (1,3- dimethylbutyls)-N '-diphenyl-para-phenylene diamines) Department)
Wax：SUNNOC waxes (the emerging chemical industrial company of imperial palace)
Sulphur：Powder sulphur (He Jian chemical companies)
Vulcanization accelerator NS：(the emerging chemical industry of imperial palace is public by NOCCELER NS (N tert butyl benzothiazole 2 sulfenamide) Department)
Silica：Nipsil AQ (wet silica) (Japanese silica company)
Silane coupling agent：Si266 (bis- (3- triethoxysilylpropyltetrasulfides) disulphide) (goldschmidt chemical corporation)
Accelerator D PG：NOCCELER D (N, N- diphenylguanidine) (the emerging chemical industrial company of imperial palace)
Formula according to table 13 to 16 is kneaded using 1.7L banbury mixers other than sulphur and vulcanization accelerator Material obtains mixture.Then, mix sulphur and vulcanization accelerator in the mixture, are used in combination open roll to be kneaded, obtain not Vulcanized rubber composition.Using steam vulcanizing pree in pressure 80kgf/cm2, at 150 DEG C to the unvulcanized rubber composition Vulcanization 30 minutes is carried out, vulcanized rubber composition is prepared.
Following evaluations are carried out to the vulcanized rubber composition thus prepared.As a result as shown in table 13 to 16.The benchmark of table 13 Formula is the formula of comparative example 1, and the benchmark formula of table 14 is the formula of comparative example 2, and the benchmark formula of table 15 is matching for comparative example 3 Side, the benchmark formula of table 16 are the formula of comparative example 4.
(test for viscoelastic)
Using viscoplasticity spectrometer (this making of rock institute), in 70 DEG C, strain 2%, the condition of (initial stage elongation) carries out tan δ's It measures.With the tan δ of benchmark formula for 100, by its numerical value with exponential representation.Index is higher, and heat is bigger.Index is 100 or less Formula be considered as and improve heat resistance (low-heat-generation).In other words, index is lower, and low-heat-generation is better.
(blue Berne abrasion test)
Using blue Berne abrasiometer (this making of rock institute) and in 15g/ minutes loading 3kg, slip rate 40% and Sha Su items Under part, implement abrasion test.The shape of sample is thickness 5mm, diameter 50mm.The use of grinding stone is the GC abrasive grains of granularity #80.With base The tan δ of quasi- formula are 100 (benchmark), by test result with exponential representation.The index is higher, and wearability is better.Index is more than 100 formula, being considered as improves wearability.
(tension test)
According to JIS K6251 " the determination method of rubber, vulcanized rubber or thermoplastic elastomer-tensile stress and emergent property ", use No. 3 dumbbell shape samples being made of above-mentioned vulcanized rubber piece implement tension test, with measure fracture tensile strength (TB) (MPa), Elongation at break (EB) (%).If the rubber composition by elongation at break less than 480% is used for giant tire, easy to produce Rubbery chip, thus need to improve.Further, since low fracture tensile strength can cause tire to destroy, it is therefore desirable to prevent because of material The decline of fracture tensile strength caused by the change of material.
Table 13 and 15 show, using the present invention isoprene oligomer embodiment show outstanding low-heat-generation, Wearability and elongation at break.
Table 14 and 16 is shown, outstanding low-heat-generation, wear-resisting is shown using the embodiment of the polyisoprene of the present invention Property and fracture tensile strength.
(independent text of sequence table)
SEQ ID NO：1：Prepare the forward primer of variant enzyme N77A
SEQ ID NO：2：Prepare the reverse primer of variant enzyme N77A
SEQ ID NO：3：Prepare the forward primer of variant enzyme L91D
SEQ ID NO：4：Prepare the reverse primer of variant enzyme L91D
SEQ ID NO：5：The amino acid sequence of variant enzyme N77A
SEQ ID NO：6：The amino acid sequence of variant enzyme L91D
Sequence table
<110>Sumitomo Rubber Industrie. Ltd (SUMITOMO RUBBER INDUSTRIES, LTD.)
Univ Yamagata (YAMAGATA UNIVERSITY)
<120>Isoprene oligomer and the method for manufacturing isoprene oligomer
<140> 2013800452905
<141> 2013-09-02
<150> JP2012-200884
<151> 2012-09-12
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<220>
<221> primer_bind
<223>Prepare the forward primer of 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>Prepare the reverse primer of 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>Prepare the forward primer of 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>Prepare the reverse primer of 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>The 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>The 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. a kind of isoprene oligomer, which is characterized in that the allyl diphosphonic acid represented by lower formula (X) and following formula (Y) institute The compound synthesis of expression and obtain, on the allyl diphosphonic acid quantity of the isoprene unit of addition be 1 to 30,
In formula (X), n indicates that integer of 1 to 10, part isoprene unit can be modified；
In formula (Y), R indicates nitrogen-containing group, oxygen-containing group, sulfur-containing group or silicon-containing group.
2. isoprene oligomer according to claim 1, allyl diphosphonic acid, institute represented by the formula (X) The compound and prenyl diphosphate for stating formula (Y) expression are synthesized into.
3. isoprene oligomer according to claim 1 or 2, under the allyl diphosphonic acid represented by the formula (X) is Allyl diphosphonic acid represented by formula (X-1), at least one atom or atomic group quilt contained in the parts II in following formula (X-1) Different atoms or atomic group replace, and atom or atomic group be not by different atoms contained in the parts III in following formula (X-1) Or atomic group substitution,
In formula (X-1), n indicates integer of 1 to 10.
4. isoprene oligomer according to claim 1 or 2, the synthesis is lived using with prenyltransferases Property enzyme carry out.
5. a kind of isoprene oligomer, which is characterized in that the isoprene oligomer is represented by following formula (Z-1) or under Formula (Z-2) is represented, and at least one atom or atomic group contained in the parts v in following formula (Z-1) or following formula (Z-2) are by nitrogen original Son, oxygen atom, sulphur atom, silicon atom, nitrogen-containing group, oxygen-containing group, sulfur-containing group or silicon-containing group substitution,
In formula (Z-1), n indicates integer of 1 to 10；M indicates 1 to 30 integer；Y indicates hydroxyl, formoxyl, carboxyl, alkoxy Carboxyl, alkoxy carbonyl or OPP bases；At least one atom or atomic group contained in the parts iv can be by different atoms or atom Group's substitution；
In formula (Z-2), n indicates integer of 1 to 10；M indicates 1 to 30 integer；Y indicates hydroxyl, formoxyl, carboxyl, alkoxy Carboxyl, alkoxy carbonyl or OPP bases；At least one atom or atomic group contained in the parts iv can be by different atoms or atom Group's substitution.
6. in the parts iv in isoprene oligomer according to claim 5, the formula (Z-1) or the formula (Z-2) Contained at least one atom or atomic group is replaced by different atom or atomic group.
7. a kind of manufacturing method of isoprene oligomer, which is characterized in that the isoprene oligomer is by lower formula (X) institute table The compound synthesis represented by allyl diphosphonic acid and following formula (Y) shown, the isoprene of addition on the allyl diphosphonic acid The quantity of unit is 1~30,
In formula (X), n indicates that integer of 1 to 10, part isoprene unit can be modified；
In formula (Y), R indicates nitrogen-containing group, oxygen-containing group, sulfur-containing group or silicon-containing group.
8. the manufacturing method of isoprene oligomer according to claim 7, the isoprene oligomer is by the formula (X) compound and prenyl diphosphate synthesis that allyl diphosphonic acid, the formula (Y) represented by indicate.
9. the manufacturing method of isoprene oligomer according to claim 7 or 8, the allyl represented by the formula (X) Diphosphonic acid is the allyl diphosphonic acid represented by following formula (X-1), at least one atom contained in the parts II in following formula (X-1) Or atomic group is replaced by different atom or atomic group, atom or atomic group contained in the parts III in following formula (X-1) not by Different atoms or atomic group substitution
In formula (X-1), n indicates integer of 1 to 10.
10. the manufacturing method of isoprene oligomer according to claim 7 or 8, the synthesis is used with isoamyl two The enzyme of alkenyl transferase active carries out.
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