REVIEW OF NATURAL RUBBER BIOSYNTHESIS AND
SYNTHESIS OF MODEL INTERMEDIATES FOR THE PREPARATION OF
A MACROINITIATOR FOR THE IN VITRO SYNTHESIS OF
POLYISOBUTYLENE-POLYISOPRENE DIBLOCK COPOLYMER
A Thesis
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Emilie Gautriaud
December, 2006 REVIEW OF NATURAL RUBBER BIOSYNTHESIS AND
SYNTHESIS OF MODEL INTERMEDIATES FOR THE PREPARATION OF
A MACROINITIATOR FOR THE IN VITRO SYNTHESIS OF
POLYISOBUTYLENE-POLYISOPRENE DIBLOCK COPOLYMER
Emilie Gautriaud
Thesis
Approved: Accepted:
______Advisor Dean of the College Dr. Judit E. Puskas Dr. Frank N. Kelley
______Faculty Reader Dean of the Graduate School Dr. Coleen Pugh Dr. George R. Newkome
______Department Chair Date Dr. Mark D. Foster
ii
ABSTRACT
A detailed review of the biochemical literature concerning the biosynthesis of polyisoprenes, including natural rubber (NR, cis-1,4-polyisoprene cPIP) by rubber producing plants, and the polymer chemical literature on biomimetic and related syntheses, was made. It led us to postulate that the biosynthesis of polyisoprenoids in general, and that of NR in particular, may proceed by a living carbocationic polymerization process. Our analysis led to the formulation of a “natural living carbocationic polymerization” (NLCP) mechanism in terms of accepted polymer chemical formalism, i.e., initiation, propagation, and equilibria between active and dormant species. A thorough analysis of the intermediates known to be involved in the biosynthesis of NR is consistent with our postulate.
This review of the biochemical literature also inspired the concept of producing new block copolymers utilizing this process and a synthetic macroinitiator. A feasible synthetic pathway was developed to yield the target macroinitiator by combining conventional syntheses and natural rubber biosynthesis. This macroinitiator will consist of a polyisobutylene (PIB) chain carrying a neryl pyrophosphate functional group (PIB-
NPP) that will be synthesized from PIB-OH made by “traditional” living carbocationic polymerization. The ultimate purpose is to explore if the cis-prenyltransferase enzyme
iii
involved in NR biosynthesis would recognize this new synthetic molecule as an initiator
of cis-1,4-polyisoprene, such that this synthetic PIB-NPP macroinitiator would induce
NR biosynthesis in vitro, and if so, could be used to produce PIB-b-NR block copolymer.
A six-step synthetic strategy for the preparation of the PIB-NPP macroinitiator was proposed. The first three steps were successfully completed. The others were studied
by carrying out experiments using model compounds, and the results obtained were
extremely promising. In particular, a model molecule was successfully linked to the PIB-
OH starting material.
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TABLE OF CONTENTS
Page
LIST OF TABLES...…………………………………………………………….………..ix
LIST OF FIGURES……………………………………………………………..………...x
CHAPTER
I. INTRODUCTION………………………………………………….…………………...1
II. BACKGROUND……………………….………………………………………………3
2.1. General Information………………….…………….…………………...…….5
2.2. Structure of Natural Rubber…...………………….………………….……….7
2.3. Synthetic cis-Polyisoprene………...…………...…………………..…………9
2.4. Alternative Sources of Natural Rubber……...….……...……………………12
2.5. In vivo Natural Rubber Biosynthesis.….……….……………...……………14
2.5.1. “Biochemical” General Mechanism……..………..………………14
2.5.2. Biosynthesis of the Isopentenyl Pyrophosphate Precursor…..…....16
2.5.2.1. The Mevalonate Pathway………….…………….………17
2.5.2.2. The Non-Mevalonate Pathway………….………………21
2.5.3. Biosynthesis of Allylic Diphosphate Cosubstrates..……...……….22
2.5.4. Role of the Activity Cofactors……………………………...……..24
2.5.5. Chemical Mechanism of Natural Rubber Biosynthesis……...……24
2.6. In vitro Natural Rubber Biosynthesis.….……...……………………………29
v
III. EXPERIMENTAL…………………………………………………………...………32
3.1. Materials (Reagents and Solvents)..…………………………………………32
3.2. Procedures………………....……………………………………………...…33
3.2.1. Handling of Air Sensitive Materials………………………………33
3.2.1.1. Vacuum Apparatus……………….……...………………33
3.2.1.2. Inert Atmosphere Dry Box………………………………34
3.2.2. Thin Layer Chromatography (TLC)………..……..………………34
3.2.3. Flash Column Chromatography……………………...……………35
3.2.4. Nuclear Magnetic Resonance (NMR) Spectroscopy…...…………36
3.2.5. Size Exclusion Chromatography (SEC)…..……………………….36
3.2.6. Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Masse Spectrometry (MS)………………………..37
3.3. Syntheses…………………………………………………….………………37
3.3.1. Synthesis of 3,7-dimethyl-1-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadiene (PN, product 2) - Step 1…………...……………37
3.3.2. Synthesis of 2,6-dimethyl-8-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadien-1-ol (PN-AOH, product 3) - Step 2……….…..…38
3.3.3. Synthesis of 1-bromo-2,6-dimethyl-8-[(tetrahydro-2H-pyran- 2-yl)oxy]-(E)-2,6-octadiene (PN-ABr, product 4) – Step 3….……39
3.3.4. Reaction of PN-ABr and 2-phenyl-1-propanol Using an Alkali Metal Carbonate……………………………………………39
3.3.5. Reaction of 3-bromo-2-methylpropene and 2-phenyl- 1-propanol Using Sodium Hydride……………..…………………40
3.3.6. Reaction of PIB-OH and 3-bromo-2-methylpropene Using Sodium Hydride……………………………………………40
3.3.7. Synthesis of 2-methyl-2-propene p-toluenesulfonate Model Compound M-OTs Using Triethylamine…………...……..41
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3.3.8. Synthesis of 2-methyl-2-propene p-toluenesulfonate Model Compound M-OTs Using Sodium Hydride………...……..41
3.3.9. Synthesis of PIB-O-M……………………………….……………42
3.3.10. Bromination of 3-methyl-2-buten-1-ol…………..…..…………..43
3.3.11. Synthesis of Tris(tetra-n-butylammonium) Hydrogen Pyrophosphate [(NBu4)3HP2O7]……..……….……….…………43
IV. RESULTS AND DISCUSSION……………………………………..………………44
4.1. Natural Rubber Biosynthesis: A Living Carbocationic Polymerization?...... 44
4.1.1. The Mechanism of Natural Rubber Biosynthesis: A Living Carbocationic Polymerization?…………..………………..………44
4.1.1.1. Initiation…………………………………………………45
4.1.1.2. Propagation………………………...……………………48
4.1.1.3. Termination………………...……………………………52
4.1.2. Conclusion: Natural Living Carbocationic Polymerization (NLCP)……..…………………………………...…………………54
4.2. Synthesis of Model Intermediates for the Preparation of Polyisobutylene-Neryl Pyrophosphate (PIB-NPP) Macroinitiator…….……57
4.2.1. Syntheses of the Protected Nerol PN (step 1) and the Allylic Alcohol PN-AOH (step 2)………….………..……60
4.2.2. Synthesis of the Allylic Bromide PN-ABr (step 3)…….…………66
4.2.3. Model Reactions for the Synthesis of PIB-PN (product 5)…..……72
4.2.3.1. Reaction of PN-ABr and an Alcohol Model Compound Using Alkali Metal Carbonate………...……72
4.2.3.2. Reaction of a Brominated and a Hydroxyl Model Compound Using Sodium Hydride…………...…73
4.2.3.3. Reaction of PIB-OH and a Brominated Model Compound Using Sodium Hydride…………...…77
vii
4.2.3.4. Tosylation of PN-AOH………………….………………77
4.2.3.5. Synthesis of PIB-O-M…………………………………..80
4.2.4. Preliminary Model Experiments for the Synthesis of PIB-NPP from PIB-N………….…………...….……….………94
4.2.4.1. Bromination of the Hydroxyl Group of a Model Compound Standing for PIB-N……………..…95
4.2.4.2. Synthesis of Tris(tetra-n-butylammonium) Hydrogen Pyrophosphate………………………..………98
V. CONCLUSION………………………………………………….……..……………100
REFERENCES……………..……………………………….……………….…………102
APPENDICES……………………………………………….…………………………119
APPENDIX A. STRUCTURE OF THE ADENOSINE TRIPHOSPHATE MOLECULE………………………………………………..….…….120
APPENDIX B. STRUCTURE OF THE NADP+ MOLECULE……………...………121
APPENDIX C. NMR SPECTRA OF PIB-OH ETHERIFIED WITH A METHYL PROPENE MODEL COMPOUND…………..………….122
APPENDIX D. NMR SPECTRA OF TRIS(TETRA-N-BUTYLAMMONIUM) HYDROGEN PYROPHOSPHATE…………………………………123
viii
LIST OF TABLES Table Page
4.1. Chen experimental conditions for the synthesis of a pyrophosphate molecule….94
4.2. Coates experimental conditions for the synthesis of geranyl pyrophosphate……95
ix
LIST OF FIGURES Figure Page
2.1. Visualization of Yokozawa’s concept of chain-growth polycondensation………..4
2.2. Attempted “bio-inspired” synthesis of cis-1,4-polyisoprene…………...…………5
2.3. The microstructure of natural Hevea rubber………………………………………7
2.4. Possible enchainments in polyisoprene……………………………………...……8
2.5. Structure of isopentenyl pyrophosphate IPP at pH = 7……………………..……15
2.6. Natural rubber biosynthesis…………………………...…………………………16
2.7. Paramagnetic (a) and diamagnetic (b) mechanisms of acetyl-CoA synthesis by CODH/ACS………………………………………………..…………………19
2.8. The mevalonate pathway to isopentenyl pyrophosphate………………...………20
2.9. Deoxy-xylulose phosphate pathway of IPP (8) and DMAPP (9) biosynthesis….21
2.10. Initiator formation in natural rubber biosynthesis……………….………………22
2.11. Structure of the allylic oligoisoprene pyrophosphates (APPs)…………..………23
2.12. Natural rubber biosynthesis with polymer chemical symbolism………...………25
2.13. Mechanism of polyisoprene formation proposed by Archer………….…………26
2.14. “Ionization-Condensation-Elimination” mechanism proposed by Poulter et al…27
2.15. Comparison of the effectiveness of NPP (∆) and GPP (∇) in the in vitro conversion of 14C-IPP. (Ο) control………………………………………………29
2.16. Effect of chain length on the rate of IPP incorporation into enzymatically active rubber particles isolated from Parthenium argentatum……..……………30
4.1. Proposed initiation mechanism in natural rubber biosynthesis (En = enzyme)…………………………………………...………………………46 x
4.2. Possible role of divalent cations in natural rubber biosynthesis…………………47
4.3. Isomers obtained by proton loss from a tertiary carbocation……………………49
4.4. Visualization of the natural rubber synthesis in vivo…………………….………50
4.5. A scheme of the active sites in avian trans-prenyltransferase. DDxxD –Aspartic acid rich motifs; the oval represents a large amino acid proposed to stop chain growth……………………...……………………………51
4.6. Termination: hydrolysis of the chain end………………………………..………53
4.7. Proposed natural living carbocationic polymerization mechanism ……..………55
4.8. Synthetic strategy to produce the PIB-NPP macroinitiator………...……………58
4.9. 1H NMR spectrum of nerol (N, product 1)………………………………………59
4.10. 1H NMR spectrum of 3,7-dimethyl-1-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadiene (PN, product 2)…………………………………….…………61
4.11. 13C NMR spectrum of 3,7-dimethyl-1-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadiene (PN, product 2)……………………….………………………63
4.12. 1H NMR spectrum of 2,6-dimethyl-8-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadien-1-ol (PN-AOH, product 3)………………….…………………64
4.13. 13C NMR spectrum of 2,6-dimethyl-8-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadien-1-ol (PN-AOH, product 3)………………….…………………65
4.14. 1H NMR spectrum of 1-bromo-2,6-dimethyl-8-[(tetrahydro-2H-pyran- 2-yl)oxy]-(Z)-2,6-octadiene (product 4)…………………………………………67
4.15. 13C NMR spectrum of 1-bromo-2,6-dimethyl-8-[(tetrahydro-2H-pyran- 2-yl)oxy]-(Z)-2,6-octadiene (product 4)…………………………………………68
4.16. Structure of 2-methyl-2-propen-1-ol………………………………..……………69
4.17. 1H NMR spectrum of the brominated model alcohol……………………………70
4.18. 13C NMR spectrum of the brominated model alcohol……………...……………71
4.19. Structure of 2-phenyl-1-propanol……………………………………..…………72
4.20. Structure of 3-bromo-2-methylpropene……………………….…………………73
xi
4.21. 1H NMR spectrum of the ether model compound………….……………………75
4.22. 13C NMR spectrum of the ether model compound………………………………76
4.23. Pathway for the synthesis of PIB-PN with model compounds involving a tosylate intermediate.…………………………………………..……77
4.24. 1H NMR spectrum of 2-methyl-2-propene p-toluenesulfonate M-OTs……….…79
4.25. 13C NMR spectrum of 2-methyl-2-propene p-toluenesulfonate M-OTs…...….…80
4.26. 1H NMR spectrum of PIB-OH…………….……………………………………..81
4.27. 13C NMR spectrum of PIB-OH……...…….……………………………………..83
4.28. SEC traces (light scattering and differential refractometer detectors) of PIB-OH……………………………….……………………………….………84
4.29. MALDI-TOF MS spectrum of PIB-OH cationized with silver ions……….……86
4.30. An isotope cluster in the MALDI-TOF MS spectrum of PIB-OH……...……….87
4.31. Structure of the PIB-OH molecule identified using MALDI-TOF MS….………88
4.32. 1H NMR spectrum of PIB-O-M…..……….……………………………………..89
4.33. 13C NMR spectrum of PIB-O-M…..……….…...………………………………..90
4.34. SEC traces (light scattering and differential refractometer detectors) of PIB-O-M………………...…………………………………………………….91
4.35. MALDI-TOF MS spectrum of PIB-O-M cationized with silver ions……..……92
4.36. Representation of the PIB-O-M 20-mer………………………..……………….93
4.37. Structure of the PIB-O-M molecule identified using MALDI-TOF MS…….…..93
4.38. Structure of 3-methyl-2-buten-1-ol………………………………………………95
4.39. 1H NMR spectrum of the brominated model intermediate of Chen’s procedure……………………………………………………………...96
4.40. 13C NMR spectrum of the brominated model intermediate of Chen’s procedure……………………………………………………………...97
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CHAPTER I
INTRODUCTION
Polyisoprenoids represent one typical class of biopolymers produced by living organisms and exhibit unique structures and features. Natural rubber is one example of polyisoprenoids and one of the most familiar and easily available biopolymers. Because of its valuable characteristics such as elasticity, natural rubber is the most abundantly used biopolymer, together with cellulose, lignin and starch. Microbiologists, biochemists and molecular geneticists have extensively studied its structure, biosynthesis, and degradation since the end of the 19th century and efforts are made to improve its production.
While some of the basic steps of natural rubber biosynthesis have been elucidated from the biochemical point of view, our understanding of this process in terms of synthetic polymer chemistry is practically nonexistent. Our objective was to carry out an
extensive study of the chemical mechanism of NR biosynthesis. This led us to propose
that this natural process is consistent with the mechanism of living/controlled
polymerizations.
1
Another objective was to synthesize a polyisobutylene-based macroinitiator that
would be used later on to study modified in vitro biosynthesis processes. Combination of fully-synthetic living carbocationic polymerization, yielding the precursor of the macroinitiator, with NR biosynthesis, would produce a fundamentally novel block copolymer: polyisobutylene-b-cis-1,4-polyisoprene (PIB-b-NR). We proposed a six-step pathway for the preparation of the PIB-NPP macroinitiator and conducted experiments using the desired materials as well as model molecules to successfully synthesize novel intermediates compounds.
2
CHAPTER II
BACKGROUND
De la Torre and Sierra1 quoted: “The appealing beauty of the routes that Nature uses to build natural products is breathtaking and the quest for laboratory syntheses that mimick these routes is longstanding.” The exponential rise in the syntheses of “bio- inspired” polymers, and the use of enzymes to mediate organic reactions powerfully underlines this view.2-9 Enzymatic catalysis in vitro, i.e., under simulated physiological conditions, leads to products identical to “natural” (in vivo) compounds, or to an
“artificial” version of the natural product.10-12 Bacterial enzymes have been used to synthesize polyesters in vitro,13-18 and a variety of isolated enzymes have been used both in vitro and under “artificial” conditions.15,19-40 Further recent examples are the syntheses of functionalized amphiphilic polymers and polyesters, and the ring-opening polymerization of seven-membered ring lactones catalyzed by immobilized Candida
Antarctica.41-43 Enzymatic catalysis has also been used to initiate polymerizations from surfaces.44-47
The selectivity and efficiency of enzymes render them attractive catalysts. The study of natural and biomimetic organic syntheses may yield non-enzymatic processes, and “synthetic methodology inspired by biogenesis”.1,48-52
3
Yokozawa et al.’s48-49 recent work is particularly revealing in this context.
Inspired by the biosynthesis of many natural biopolymers, these authors developed the
concept of “chain-growth polycondensation”, according to which an enzyme activates the
initiating entity and/or the dormant polymer chain end, which proceeds to add monomer
and relinquishes the protective end group. Figure 2.1 helps to visualize this concept.
ACTIVE INACTIVE ACTIVE + Initiator Monomer
INACTIVE ACTIVE
Figure 2.1. Visualization of Yokozawa’s concept of chain-growth polycondensation.48-49
Examples cited by these authors include peptide extension (termed “elongation” in
biochemistry),53,54 DNA55 and RNA56 syntheses and natural rubber biosynthesis.57-59
Yokozawa’s group developed two strategies for chain-growth polycondensation. The first strategy involved the activation of polymer end groups by substituents, and led to aromatic polyamides, polyesters, polyethers, poly(ether sulfone) and polythiophene. By the second strategy the monomer was separated from the polymerization phase to prevent monomer-monomer and polymer-polymer condensations. Yokozawa’s work, while a breakthrough in bio-mimetic polymer synthesis, did not produce the desired monodisperse polypeptides or DNA. The synthesis of cPIP was attempted by the use of
4
an electrophilic initiator by the pathway shown in Figure 2.2; however, only a diene was
obtained.48
R R R R R
El El 1 El El X X X X - SiMe3X - SiMe3 REACTIVE n SiMe3 UNREACTIVE 1 - SiMe3X R El = electrophilic initiator
Figure 2.2. Attempted “bio-inspired” synthesis of cis-1,4-polyisoprene.48
The proposed chain-growth polycondensation mechanism should lead to monodisperse
cPIP; however, all natural rubbers exhibit multimodal/broad molecular weight
distributions.60-61
The objective of this chapter is to critically review research aimed at the synthesis
of NR. Insight generated by this review was used to develop a proposition for the
mechanism of NR biosynthesis (see CHAPTER IV).
2.1. General Information
NR, arguably the most important polymer produced by plants, is a strategically
important raw material used in many thousands of products, including hundreds of
medical devices. NR is obtained from latex, an aqueous emulsion present in the
laticiferous vessels (ducts) or parenchymal (single) cells of rubber-producing plants.
Although more than 2,500 plant species are known to produce NR, currently there is only
5
one important commercial source, Hevea brasiliensis (the Brazilian rubber tree). The
rubber from Parthenium argentatum, also called guayule, is being marketed as “non-
allergenic natural rubber”.60-63 We do not know why plants produce rubber and our
understanding of the mechanism of NR biosynthesis is far from complete.
The rubber latex from H. brasiliensis is harvested by “tapping” the rubber tree,
i.e., making an incision in the trunk and collecting the sap freely oozing out of the ducts.
The raw polymer is recovered from the latex by coagulation and drying, yielding high
molecular weight (>1 million g/mol) “crepe”.
While Charles Goodyear is credited with discovering the crosslinking of NR by sulfur in 1839, ancient Mesoamerican peoples discovered the advantages of crosslinking much earlier: they mixed the rubber latex harvested from the Castilla elastica tree with the juice of Ipomoea alba (a species of morning glory vine) and produced solid rubber.
Recent analysis of Olmec rubber balls (1600-1200 B.C.) could not identify the exact chemical nature of the crosslinks, although the dynamic mechanical properties of the rubber crosslinked by the ancient method closely resembled those of modern vulcanized
NR.64 Vulcanized NR exhibits an excellent combination of properties, including
elasticity, resilience, abrasion resistance, efficient heat dispersion and impact resistance,
which led to sustained research and development efforts to develop synthetic processes to
produce NR. To date, this objective has not been achieved.
6
2.2. Structure of Natural Rubber
Despite extensive research, the exact structure of NR is still unknown. Early X-
ray diffraction studies showed that the double bonds of the isoprene repeat units are in cis
configuration.65 By the use of 1H and 13C-NMR spectroscopies, Tanaka et al.66 later showed that the second and third units of Hevea rubber are trans, followed by repetitive cis enchainment (Figure 2.3). The terminal groups are believed to be -CH2OH or a fatty
acid ester.
Figure 2.3. The microstructure of natural Hevea rubber.66
The presence of cyclized polyisoprene sequences in NR was also detected. Other chemical groups, termed “abnormal” were also identified (aldehydes, epoxides and amines); however, their origin remains unknown.
5 6 H. brasiliensis and P. argentatum produce high molecular weight (Mn = 10 –10
60,61,67-69 g/mol) and broad/bimodal molecular weight distribution (Mw/Mn = 2-10) NR. Mn stands for number average molecular weight and Mw stands for weight average molecular weight. Both rubbers contain gelled (crosslinked) fractions, but it is not known if gelation
7
occurs in the plants or arises during processing. The chemical structure of natural cPIP
rubbers obtained from different plant species (Russian dandelion, goldenrod, Jelutong,
etc.) differs only in the number of the initial trans units (0 to 3) following the 1,1-
dimethylallyl head group. Only a relatively few species, such as balata and Gutta percha,
produce gutta or trans-1,4-PIP. Chicle produces a 1/4 mixture of cPIP and trPIP.60,61
The different structures of possible enchainments in polyisoprene are shown in
Figure 2.4.
Figure 2.4. Possible enchainments in polyisoprene.
8
2.3. Synthetic cis-Polyisoprene
At the present no synthetic cPIP is able to mimic the performance of NR.70,71 Even
synthetic high-cis-polyisoprene has inferior properties (e.g., “green” tensile strength and
tack).72 The main NR properties remaining unmatched are its defect-free microstructure
(100% cis-1,4-polyisoprene with monomeric units linked regularly in a head-to-tail
fashion), its special unique chain defects due to heteroatoms (aldehydes, ketones…) or
proteins, and its ability to undergo strain-induced crystallization.73 This renders natural
rubber an essential renewable resource material.
Efforts have been made by generations of polymer scientists to produce high cis-
1,4-polyisoprene in laboratory and on industrial scale. Lanthanide-based polymerization
of isoprene is one of the bests methods to make >98% cis-1,4-polyisoprene.74-85
Neodynium-based catalysts activated by aluminum alkyls (AlR3) display high cis-1,4
stereospecificity in isoprene polymerization; however the molecular weight and
molecular weight distribution of the resulting polymers are not well controlled, which is
attributable to the heterogeneity of the catalyst systems and the multiple nature of the
active centers. Thus these PIPs do not match the properties of NR.
In 2002, Dong and Masuda used a new catalyst composed of neodymium (III)
isopropoxide [Nd(OiPr)3] and methylaluminoxane (MAO) to synthesize PIP with 90-92%
cis-enchainments.86 In 2003, they improved the cis-1,4 stereospecificity up to ~94% by
adding tert-butyl chloride to this same catalyst.87,88 The effect of chloride on the success
9
of the polymerization was evidenced earlier, as Evans and Giarikos pointed out in their
recent comparative study.89
Russian scientists developed a neodynium-based high cis-1,4-polyisoprene having
98.5% cis content and 1.5% 3,4 units, which was claimed to be free from 1,1- (head-to-
head type) and 4,4-regiodefects (tail-to-tail type), as well as trans units.90 Halasa et al.91 recently patented an even more efficient neodymium catalyst system that can be used to produce synthetic PIP rubber having a very high cis-microstructure content (98.0% to
99.5%) and high stereoregularity. This catalyst system is prepared by reacting a neodymium carboxylate with an organoaluminum compound, and by reacting then the neodymium-aluminum catalyst component obtained with an elemental halogen. The inventors claim that the synthetic PIP rubber produced crystallizes under strain and can be compounded into rubber formulations in a manner similar to NR. They obtained PIP with Mn similar to NR (100,000 to 1,500,000 g/mol) and molecular weight distribution
Mw/Mn from 1.0 to 2.5, i.e., narrower than NR.
Although neodymium has traditionally been the lanthanide chosen for diene
polymerizations,92 recent studies have shown that a wider variety of lanthanides can lead
to high cis-1,4-polymerization.79,80,84,85
In the case of anionic polymerization promoted by lithium initiators, the so-called
“low-cis” anionic PIP still consists of 90-92% cis-enchainments.93,94 The remaining 8-
10% is composed of trans-1,4-units, 1,2-/3,4-units, and head-to-head (1,1-) or tail-to-tail
(4,4-) enchainments of monomeric units. Later anionic polymerization initiated by lithium species produced up to 96% cis-content with 4% 3,4-vinyl enchainment.95,96 PIP
10
with very high cis-content (97%) is produced by titanium-based Ziegler-Natta polymerization, but still defects in regioselectivity are present.97
Metallocene-catalyzed IP polymerizations are also mentioned in the literature,
although dienes are believed to be poisons for these catalysts.71 In 2004 Kaita et al. were
able to obtain a PIP with almost perfect 1,4-cis microstructure (>99.99%) at 0°C in 55%
yield after 5h reaction catalyzed by a gadolinium metallocene-based complex.98 The
molecular weight of this synthetic PIP was Mn ≈ 700,000 g/mol and the molecular weight
distribution Mw/Mn = 2.01. However the authors did not specify anything about the
properties of this cPIP.
According to our extensive literature survey, 100% cPIP has never been produced
synthetically. The outstanding overall properties of NR are mainly due to the 100% cis
microstructure, together with high molecular weights and broad molecular weight
distributions of the cPIP, and the presence of proteins and the so-called “abnormal”
groups.60,61,66 Thus the synthesis of 100% cPIP using a synthetic process analogous to
natural rubber biosynthesis would be of considerable fundamental and practical
significance.
The in vitro biosynthesis of NR has been demonstrated68,69,99 (see 2.6. In vitro
Natural Rubber Biosynthesis), however only at the mg scale. The elucidation of NR
biosynthesis in terms of polymer chemical principles may lead to a synthetic strategy for
100% cPIP.
11
2.4. Alternative Sources of Natural Rubber
There is a steady increase in the demand for NR, both for its unique properties but also for the increase in demand and production of rubber goods, especially from the developing countries.73 Global dependence on the single species of Hevea Brasiliensis as a natural rubber source is quite risky - Guayule production is still limited. Indeed, H.
Brasiliensis crops have very little genetic variability, leaving rubber plantations at risk of serious pathogenic attacks. Also, experts who analyze the Asian market from where essentially all of the NR produced worldwide originates, predict that in the future instead of NR, other more profitable crops will be produced. For instance, in the 1990s Malaysia was the largest commercial NR producer in the world but has lost its leadership because of the trend to replace NR cultivation with palm oil plantations.73 In addition, repeated exposure to residual proteins in latex products based on H. brasiliensis led to serious and widespread allergenic (Type I) hypersensitivity.100-108 The quantity of potential allergens in a final product can be greatly affected by the degree of latex purification. However, patients having severe allergic reactions to Hevea latex products continue to react even upon contact with a highly purified product.73 Thus there is great interest in alternative sources of NR in the biochemistry community.109
The only alternative plant species under cultivation is Parthenium argentatum
Gray, also called guayule. Guayule rubber is synthesized in the single, thin-walled parenchyma cells of the plant’s branches, stems and roots, and thus requires a special extraction procedure to produce rubber-containing latex.73 Also, its purification is more
12
complicated, time consuming and expensive than the preparation of NR from Hevea.73
However, it is fully equivalent to H. Brasiliensis-based rubber from the chemical standpoint (cis-1,4-polyisoprene in both cases) and shows comparable molecular weights
73 (Mn and Mw) if properly purified. Thus guayule rubber has properties close to those of
Hevea rubber although its mechanical properties remain somewhat inferior.110,111 In terms of processability, it is better because it is softer, and it has lower (Mooney) viscosity.73
Guayule rubber also reportedly shows a better tendency to undergo strain-induced crystallization than Hevea rubber.112,113
Active research and development programs are underway to domesticate and commercialize guayule already marketed as “non-allergenic natural rubber”.60,61,63,114-116
P. argentatum is suitable for the manufacture of high-quality medical products.117,118
Factors affecting the extractability and stability of latex rubber in harvested guayule shrub are under investigation to maximize latex yields, and optimize post-harvest practices.119-121 In order to further increase yields in guayule, studies are beginning to
elucidate the biochemical regulation of rubber yield (principally rate of synthesis) and
quality (principally molecular weight distribution) at the biochemical and genetics
levels.122 An alternative approach to increase rubber production is to target the rubber
biosynthesis pathway directly using recombinant DNA technology.123 The effect of three
different allylic pyrophosphate synthase transgenes placed into three P. argentatum lines,
on growth, resin and rubber production under field conditions has recently been
evaluated. Veatch et al.124 observed some differences in the growth, and an increase of
the resin content without impact on rubber levels. They conclude optimistically since
13
their studies show that transformation is possible. Other plant species do not produce the
sufficiently high molecular weights required for commercial applications.67-69
2.5. In vivo Natural Rubber Biosynthesis
Scientists have extensively studied the biosynthesis of NR. The following part
summarizes the information pertaining to the in vivo NR biosynthesis found in the
biochemical literature.
2.5.1. “Biochemical” General Mechanism
The biosynthesis of NR is catalyzed by a rubber transferase enzyme (EC 2.5.1.20,
cis-prenyl transferase66,125-130). Enzymes are proteins that act as highly selective catalysts
in biochemical reactions. Rubber transferase is integral to the monolayer membrane that
surrounds microscopic, cytosolic rubber particles. Polymerization takes place within the
boundary biomembrane monolayer between nonpolar rubber particles and the aqueous
medium.131
The phospholipid monolayer stabilizes the particles, preventing aggregation. The hydrophobic chains are segregated inside the latex particles, and polymerization proceeds
at the active sites of the enzyme.123,132 The rubber transferase enzyme is most likely
amphiphilic, with glycosylated hydrophilic regions mediating the access of the
hydrophilic building blocks and, subsequently, the hydrophobic regions mediating
placement into the biomembrane.133
14
The monomer is isopentenyl pyrophosphate IPP. Figure 2.5 shows the structure of
IPP, an adduct of pyrophosphoric acid (H4P2O7) and isoprene (IP).
CH3 OOHOOH H2 C C P P H2C C O O OH H2
IPP at pH = 7
Figure 2.5. Structure of isopentenyl pyrophosphate IPP at pH = 7.
An allylic diphosphate (dimethylallyl-pyrophosphate, DMAPP) initiates chain growth. IPP and DMAPP are termed “substrate” and “cosubstrate” respectively in the biochemical literature. These entities would be termed monomer and initiator respectively by polymer chemical terminology. Other cosubstrates/initiators include oligomers of DMAPP such as geranyl-, farnesyl- and geranyl-geranyl-pyrophosphate
(GPP, FPP, GGPP). Enzymatic activity requires the presence of divalent cations, such as
Mg2+ or Mn2+, called “activity cofactors”.125-128,134,135 The substrate, cosubstrates and cofactors are hydrophilic, and the produced rubber is hydrophobic; the amphiphilic enzyme catalyst is located at the interface between the rubber particles and the aqueous phase of the latex. Figure 2.6 shows a flow diagram of NR biosynthesis as represented, reproduced from the biochemical literature.68
15
Figure 2.6. Natural rubber biosynthesis.
In each step, one molecule of pyrophosphoric acid (HPP or its salts) is generated; i.e., the process is a combination of chain growth and polycondensation, as suggested by
Yokozawa.136,137 Molecular weights and molecular weight distributions are species-
dependent; however, the biochemical literature is mute in regard to the control of these
parameters in vivo.69 Broad/multimodal molecular weight distributions are due to
branching and/or crosslinking via acid-catalyzed cyclization, or by “abnormal” functional
groups (aldehydes, epoxides and amines).60,61,66
2.5.2. Biosynthesis of the Isopentenyl Pyrophosphate Precursor
For several decades, the mevalonate (MVA) pathway was believed to be the
unique source of isoprenoid building blocks, and the existence of a separate pathway
leading to IPP was ignored. Yet a mevalonate-independent (non-MVA) alternative
pathway (deoxy-xylulose pathway) also leads to the precursor IPP. These two distinct
16
pathways have evolved in different organisms. In eukaryotes (organisms whose cells
contain a nucleus) and archaebacteria (single-celled microorganisms similar to other
prokaryotes in most aspects of cell structure and metabolism, however genetically distinct
from them), IPP is derived from acetyl-CoA, while in bacteria (single-celled
microorganisms whose cells lack a nucleus; also called prokaryotes) and plant
chloroplasts (chlorophyll-containing organelles; an organelle is a discrete structure of a
cell having specialized functions) IPP is derived from 1-deoxy-D-xylulose-5-
phosphate.138 In higher plants, the conventional mevalonate pathway operates mainly in
the cytoplasm (substance within a biological cell that is not contained in the nucleus) and
mitochondria (organelles located in the cytoplasm and containing enzymes responsible
for the conversion of food to usable energy), whereas the non-MVA pathway operates in
the plastids (major organelles found only in plants and algae, classified as chloroplasts,
leucoplasts, amyloplasts or chromoplasts depending on their morphology and function).139 The IPP polymerized by the rubber transferase is probably derived from the
MVA pathway, also located in the cytosol.140 But some IPP produced by the 1-deoxy-D-
xylulose-5-phosphate pathway may diffuse from the plastids to the cytosol (the internal
fluid of the cell).141
2.5.2.1. The Mevalonate Pathway
The mevalonate mechanistic pathway involves the formation of acetyl coenzyme
A (acetyl-CoA) and mevalonate (MVA) as intermediates. The bifunctional nickel-iron sulfur enzyme called carbon monoxide dehydrogenase/acetyl-CoA synthase
(CODH/ACS) plays a key role in the pathway of acetyl-CoA formation. A NiFeS cluster
17
on CODH (cluster C) catalyzes the reversible reduction of CO2 to CO (equilibrium 1),
whereas a separate active site NiFeS cluster on ACS (cluster A) catalyzes acetyl-CoA
synthesis from CO, CoA, and a methylated corrinoid iron-sulfur protein (CFeSP)
(equilibrium 2).142
- + CODH CO2 + 2e + 2H CO + H2O (1)
ACS + CH3-CFeSP + CO + CoA CH3-C-SCoA + H + CFeSP (2) O acetyl-CoA
Cluster A on ACS forms a paramagnetic adduct with CO, called the nickel-iron-
carbon (NiFeC) species, which Ragsdale et al.142 have hypothesized to be a key
intermediate in acetyl-CoA synthesis. This hypothesis was controversial until Ragsdale reported the results of steady-state kinetic experiments and of kinetic simulations of the steady-state and transient kinetic that strongly support the kinetic competence of the
NiFeC species in the Wood-Ljungdahl pathway of anaerobic CO2 fixation. Figure 2.7
shows the two competing hypotheses for acetyl-CoA synthesis. In the “paramagnetic
catalytic cycle” (Figure 2.7.a), the NiFeC species is the key intermediate, whereas in the
“diamagnetic catalytic cycle” (Figure 2.7.b), it is not part of the catalytic cycle.
18
R RS S Ni 2 RS Ni 2 RS N N N
O 2 e- - O CH3 H3CCSCoA 1 e C RS CH3 RS RS Ni 2 Ni 2 Co O Ni CFeSP CoAS N RS N SR Cint Cred RS N 2 H3CCSCoA N CO Co
CoAS CO O CH3 RS RS CH3 O CH 3 Ni R C C S RS Ni2 RS N RS RS Ni 2 N 2 NiFeC Species Ni CoAS N N CH N RS N 3 Co CO CFeSP CoAS
Co O CH3 R CH3 CO Cint Cred2 CO RS C S RS RS RS Ni 2 Ni 2 Ni 3 RS Ni2 OC N H3C N H3C N N N SR SR N
(a) (b)
Figure 2.7. Paramagnetic (a) and diamagnetic (b) mechanisms of acetyl-CoA synthesis by CODH/ACS.
Mevalonate is a six-carbon intermediate in the pathway, arising from the sequential condensation of three acetyl-CoA units to generate 3-hydroxy-3- methylglutaryl Coenzyme A (HMG-CoA). HMG-CoA is converted to MVA in an irreversible reaction catalyzed by HMG-CoA reductase. MVA is then sequentially phosphorylated and decarboxylated to generate IPP by the enzymes mevalonate kinase, mevalonate 5-phosphate (MVAP) kinase, and mevalonate 5-diphosphate (MVAPP) decarboxylase, such as by adenosine triphosphate (ATP, see APPENDIX A).140 Figure
19
2.8 represents this MVA pathway.139,143,144 The structure of the NADP+ molecule is shown in APPENDIX B.
O 2 Acetyl-CoA (AC) SCoA
AAC thiolase CoASH
O O Acetoacetyl-CoA (AAC) SCoA
HMG-CoA synthase CoASH
3-Hydroxy- OOOH 3-methylglutaryl-CoA (HMG-CoA) HO SCoA
2 NADPH
+ HMG-CoA 2 NADP reductase CoASH
Mevalonate O OH (MVA) HO OH
ATP MVA kinase ADP
Mevalonate O OH phosphate (MVAP) HO OP
ATP MVAP kinase ADP
Mevalonate O OH diphosphate HO OPP (MVAPP)
ATP MVAPP ADP decarboxylase
CO2
Isopentenyl diphosphate (IPP) OPP
Figure 2.8. The mevalonate pathway to isopentenyl pyrophosphate. 20
2.5.2.2. The Non-Mevalonate Pathway
Figure 2.9 represents the non-MVA route.
O O O
COO + H O P O 1 2 OH O A CO2
OH O O O P O 3
OH O NADPH B NADP
HO O
O P O 4
OH OH O
CTP C PPi O N NH2 HO O O
O P O P O O N 5 OH OH O O
ATP HO OH D O ADP
O N NH2 O P O O O
O O P O P O O N 6 OH OH O O
HO OH E CMP
O O
O P O O P 7 O O
OH OH
O O O O F O P O P O O P O P O
O O O O 8 9
Figure 2.9. Deoxy-xylulose phosphate pathway of IPP (8) and DMAPP (9) biosynthesis.145 A: 1-deoxyxylulose 5-phosphate synthase; B: 1-deoxyxylulose 5- 21
phosphate isomeroreductase; C: 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase; D: 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; E: 2-C-methyl-D-erythritol 2,4- cyclodiphosphate synthase; F: isopentenyl diphosphate isomerase.
Briefly, 1-deoxy-D-xylulose-5-phosphate (3) obtained by condensation of
pyruvate (1) and D-glyceraldehyde 3-phosphate (2) undergoes a rearrangement coupled
to a reduction step. The resulting 2-C-methyl-D-erythritol 4-phosphate (4) is converted
into its cyclic diphosphate (7) by the sequential action of three enzymes. 2-C-Methyl-D-
erythritol 2,4-cyclodiphosphate (7) is finally transformed into IPP (8) and DMAPP (9).
2.5.3. Biosynthesis of Allylic Diphosphate Cosubstrates
IPP and its cosubstrates are associated in vivo with monovalent cations (Mt+: K+,
+ + 68,69,99 Na or NH4 ). IPP is isomerized to 1,1-dimethylallyl pyrophosphate (DMAPP) by
IPP isomerase (Figure 2.10).
OOHOOMt OOHOOMt
P P IPP isomerase P P O O O Mt O O O Mt IPP DMAPP
Figure 2.10. Initiator formation in natural rubber biosynthesis.
Catalyzed by specific trans-prenyl transferases,126 DMAPP adds 1-3 IPP units to
form oligomeric allylic pyrophosphates (APPs, Figure 2.11), all of which may function as
initiators. These APPs are geranyl-, farnesyl- and geranyl-geranyl-pyrophosphates (GPP,
FPP and GGPP respectively).
22
Monomer IPP (Substrate) (C = 5) PP
isomerization
DMAPP (C = 5) PP
+ IPP
GPP + 2 IPP (C = 10) PP + 3 IPP Initiators (Cosubstrates) FPP (C = 15) PP
GGPP (C = 20) PP
OH O Mt
where PP = O P O P O Mt O O
Figure 2.11. Structure of the allylic oligoisoprene pyrophosphates (APPs).
Soluble trans-prenyl transferases have been studied extensively, including solanesyl pyrophosphate synthase146,147 and avian trans-prenyl transferase, farnesyl
pyrophosohate synthase, which has been the model for the prenyl alkylation reaction (see
2.5.5. Chemical Mechanism of Natural Rubber Biosynthesis).138,148-157
23
2.5.4. Role of the Activity Cofactors
While the exact role of the cofactors Mg2+ and Mn2+ is still unclear, Scott et al.134 recently demonstrated that only cofactor-activated IPP monomer will interact with the enzyme, while the FPP initiator may bind even in the absence of cofactors. They also showed that the metal ion concentration in vitro can affect the rubber biosynthesis of
Ficus elastica, Hevea brasiliensis, and Parthenium argentatum: a metal ion is required for rubber biosynthesis, but an excess of metal ions interacts with the rubber transferase inhibiting its activity.134 Da Costa et al. later suggested that H. brasiliensis could use
cytosolic magnesium concentration as a regulatory mechanism for rubber biosynthesis
and molecular weight in vivo.135
2.5.5. Chemical Mechanism of Natural Rubber Biosynthesis
While the basic steps of NR biosynthesis are understood from a biochemical point
of view, our understanding of this process in terms of synthetic polymer chemical
principles is lacking. Figure 2.12 shows the biosynthesis of NR in terms of the initiation
and propagation steps proposed by Tanaka66, i.e., in polymer chemical symbolism.
24
a. Initiation
PP + PP 0 to 3 Initiator Monomer
PP + HPP
0 to 3
b. Propagation
PP + n PP 0 to 3 Monomer
cis-prenyl transferase PP + n HPP Mg2+ or Mn2+ 0 to 3 n + 1
Figure 2.12. Natural rubber biosynthesis with polymer chemical symbolism.
As mentioned earlier, one molecule of pyrophosphoric acid HPP (or PP) is generated in each step; thus the process is a combination of chain growth and polycondensation mechanisms. Figure 2.13 shows Archer et al.’s mechanism158 of
initiation in the enzymatic synthesis of cPIP.
25
H H H H
H3C C H3C C
C CH2 PP C CH2 PP
H3C H C 2 S S CH2 H C En H3C CH2 CH2 PP
En HPP
S H where stands for the sulfhydryl group H3C CH of the enzyme or a cofactor En attached to the enzyme C CH2 H3C
S CH2 C En H3C CH2 CH2 PP
Figure 2.13. Mechanism of polyisoprene formation proposed by Archer.158
According to this mechanism, first an enzyme (En) interacts with the
phosphorylated monomer IPP to form an En-IPP adduct, which reacts with another IPP while HPP is lost. The product is able to repeat (and sustain) IPP addition with the simultaneous loss of HPP. An allylic pyrophosphate initiator is not required. Originally, this mechanism was proposed as an alternative to McMullen’s theory, according to which propagation occurs by IPP-nucleotide complexes, and the nucleic acid double helix is a template for stereospecific polymerization.159 Consideration of biological precedents led
to the development of templated polymerizations proceeding with stereochemical
restrictions, and supramolecular assembly.160-165 Neither McMullen nor Archer et al.
addressed the role of the divalent cation cofactors, and both propositions remain
unsubstantiated.
26
Poulter et al. proposed the involvement of carbocationic species in prenyl transfer
reactions in natural terpenoid synthesis.138,166-168 According to these authors168 resonance-
stabilized allylic carbocationic intermediates arise during the reaction of allylic
pyrophosphate with IPP (Figure 2.14).
PP PP
R PP R PP
PP PP
H H R PP R PP
Figure 2.14. “Ionization-Condensation-Elimination” mechanism proposed by Poulter et al.168
The dissociative (SN1) mechanism is consistent with an excellent Hammet plot for rates
of solvolysis versus prenyl transfer with fluorine substituted APP. Enzyme-catalyzed
18 hydrolysis of GPP in H2 O revealed breakage of the C-O bond and inversion of chirality
at the C1 carbon.151 This mechanism is generally accepted by the biochemical
community.169
27
Polymer chemists, who may be skeptical in regard to a carbocationic
polymerization proceeding in an aqueous medium, should recall that carbocations can be
generated in aqueous media under select conditions. Thus, Sawamoto et al.170,171 described numerous cationic polymerizations in aqueous systems, and Mayr et al.172 demonstrated that carbocations can react with π nucleophiles (e.g., olefins) in aqueous media under appropriate conditions.
Chain termination is believed to occur when the enzyme relinquishes the rubber molecule; however Cornish challenged this concept.69 NR contains HO-end groups,
which are proposed to form by the hydrolytic cleavage of terminal polymer-
pyrophosphate linkages (see Figure 2.3). The chain end may also react with fatty acids,
generating ester end groups. It is not clear if this happens in vivo or during processing. At physiological pH, the pyrophosphate end group is in the dianion form and is quite stable.167 Chemical reactions occurring post-polymerization are evidenced in the
literature.60,61 Fresh latex has monomodal molecular weight distribution, while processed
Hevea rubber normally displays multimodal distribution. This may be explained by
chain-chain coupling under acidic conditions. Chain-chain coupling accompanied by
cyclization have been observed in the synthetic carbocationic polymerization of IP.173,174
Hevea rubber also has cyclized PIP sequences. It was also suggested that the “abnormal” groups form during processing.60,61 Two Hevea cis-prenyltransferase cDNAs were recently sequenced,175 but the chemistry of natural rubber biosynthesis is still
incompletely understood.
28
2.6. In vitro Natural Rubber Biosynthesis
Archer and Audley were first to initiate the in vitro synthesis of cPIP by neryl
99 14 diphosphate (NPP, C10), a cis-allylic pyrophosphate. They incubated C-IPP in the
presence of unlabelled neryl or geranyl pyrophosphate initiators in a suspension of
washed rubber particles (isolated from H. brasiliensis latex), and demonstrated that the cis-allylic neryl pyrophosphate was a more efficient initiator than the trans-allylic geranyl diphosphate (Figure 2.15).
Figure 2.15. Comparison of the effectiveness of NPP (∆) and GPP (∇) in the in vitro conversion of 14C-IPP. (Ο) control.99
The same authors found that the rate of IPP incorporation increased with the chain
length of the APP oligomer (DMAPP < GPP < FPP < GGPP). Cornish et al. found the
same trend for P. argentatum (Figure 2.16).176
29
Figure 2.16. Effect of chain length on the rate of IPP incorporation into enzymatically active rubber particles isolated from Parthenium argentatum.
Cornish’s team developed three in vitro NR synthesis systems (H. brasiliensis, P.
argentatum and Ficus elastica).69 They concluded that rubber transferases are not
particularly sensitive to the size and stereochemistry of the initiator. Similarly to in vivo
NR biosynthesis, the molecular weight and the molecular weight distribution of the
rubber depended on the species from which the living latex was harvested. This group
also showed in all three rubber-producing species that increasing the FPP initiator
concentration increased the IPP monomer incorporation rate, but decreased the polymer
molecular weight, while increasing the IPP monomer concentration increased both the
initiation and propagation rates as well as the molecular weight.68,176 Based on these
results these workers concluded that detachment of rubber molecules from the rubber transferase is not the primary regulator of molecular weight in vivo. These pioneering studies generated valuable insight into the mechanism of NR biosynthesis.
30
Natural rubber biosynthesis is an ingenious process mankind has not been able to
reproduce yet. The elucidation of the mechanism of the in vivo biosynthesis of NR is of
great theoretical and practical importance. There is surprisingly little information in the
literature about the polymer chemical aspects of NR biosynthesis, and currently no
evidence for ongoing research activity could be found in this area in the polymer
chemical community. In contrast, the biochemistry community is quite active in this area.
Pioneering in vitro studies generated valuable insight into the mechanism of NR biosynthesis. However, our literature review reinforces our belief that there is a great
need to analyze natural rubber biosynthesis from a polymer chemistry point of view.
Based on that background, we divided this thesis project into two parts. This
review of the biochemical literature led us to postulate that the biosynthesis of NR may
proceed by a living carbocationic polymerization process. Thus one target of this
Master’s work was to present a new view of NR biosynthesis from the point of view of
synthetic polymer chemistry and to conclude by proposing a mechanism for this process.
Then we wanted to verify if the cis-prenyltransferase enzyme would recognize a new synthetic molecule as an initiator of cis-1,4-polyisoprene in vitro biosynthesis. Thus the plan was to synthesize a polyisobutylene chain carrying a neryl pyrophosphate functional group (PIB-NPP) in order to explore later if this synthetic macroinitiator
would induce NR biosynthesis in vitro. If successful, PIB-b-NR diblock copolymer
would be produced.
31
CHAPTER III
EXPERIMENTAL
3.1. Materials (Reagents and Solvents)
Nerol, 3,4-dihydropyran (DHP), pyridinium p-toluenesulfonate (PPTs), tert-butyl
t hydroperoxide ( BuOOH), selenium dioxide (SeO2), salicylic acid, N-bromosuccinimide
(NBS), dimethyl sulfide ((CH3)2S), sodium hydride (NaH, dry, 95%), 3-bromo-2- methylpropene, 2-methyl-2-propen-1-ol, 2-phenyl-1-propanol, p-toluenesulfonyl chloride
(tosyl chloride), triphenylphosphine (PPh3), tetrabromomethane (CBr4), disodium
dihydrogen pyrophosphate (Na2H2P2O7), ammonium hydroxide (NH4OH, 0.104N), 40%
(w/w) aqueous tetra-n-butylammonium hydroxide (NBu4OH), 1-bromo-3-methyl-1-
butene, tris(tetra-n-butylammonium) hydrogen pyrophosphate [(NBu4)3HP2O7] were purchased from Aldrich and used as received. Molecular sieves (3Å, 8-12 mesh, Acros) were rinsed with deionized water and acetone, and then heated overnight at 180°C under vacuum. Celite was purchased from Fisher. Dowex AG 50W-X8 cation-exchange resin
+ (100-200 mesh, hydrogen form) and Dowex AG50X8 ion exchange column (NH4 form)
were obtained from Sigma. Sodium hydroxide was purchased from EMD.
32
All solvents and reagents were of reagent grade. Tetrahydrofuran (THF),
dichloromethane (CH2Cl2) and toluene were dried over calcium hydride (Aldrich) and
distilled prior to use, unless specified. Anhydrous dimethylformamide (DMF) was
purchased from Fisher Scientific in a sealed bottle and used as received. Triethylamine
(Aldrich) was dried over potassium hydroxide (EMD), distilled and stored on potassium
hydroxide prior to use. Thin layer chromatography (TLC) plates were the product of
Merck Chemical Co. GR ACS grade THF was purchased from EMD and was used for
SEC without further purification. 1,8-Dihydroxy-9(10H)-anthracenone (dithranol) and
silver trifluoroacetate (Aldrich) were used without further purification.
PIB-OH was synthesized and characterized as reported.177-180 The three samples
used for this thesis work were provided by Donna Padavan for #16 and by Yaohong Chen
for #6 and #15. Sample #16 was PIB-OH with Mn = 5600 g/mol, Mw/Mn = 1.07 and Fn =
1.21 (functionality). Sample #6 was PIB-OH with Mn = 6500 g/mol, Mw/Mn = 1.05 and
Fn = 1.09 ± 0.16 (average functionality from 24 experiments). Sample #15 was PIB-OH
with Mn = 5600 g/mol, Mw/Mn = 1.11 and Fn = 1.09 ± 0.16.
3.2. Procedures
3.2.1. Handling of Air Sensitive Materials
3.2.1.1. Vacuum Apparatus
The removal of contaminants (e.g. O2, H2O, CO2) and volatile impurities from the
glassware, solvents, and reagents used was necessary to avoid unwanted reactions for
some steps. This was accomplished by using a Schlenk line and air-free glassware. 33
The Schlenk line consisted of a 4-port single bank air-free vacuum manifold made
of Pyrex® glass tubing. All connection ports were teflon Rotaflo® stopcocks that incorporate a grease-free o-ring seal. A vacuum pump (Edwards RV8) was connected to one end of the manifold. A liquid nitrogen trap was placed between the manifold and the pump to protect the pump oil from solvent contamination. Nitrogen gas (Praxair, technical grade) was introduced into the vacuum line through the other end of the manifold. The pressure of the nitrogen gas in the vacuum line was controlled using a bubbler. Two glass drying columns packed with calcium sulfate and molecular sieves
(4Å) for one and phosphorus pentoxide for the second and placed in series between the gas cylinder and the manifold were used to dry the nitrogen gas.
3.2.1.2. Inert Atmosphere Dry Box
Sodium hydride was used for some reactions and was weighed and transferred into the air-free round bottom reaction flask inside of an inert atmosphere dry box
(Mbraun LabMaster 200B). The moisture (<1 ppm) and oxygen (<5 ppm) contents were monitored. The glassware and chemicals were placed in an antechamber that was evacuated using an Edwards vacuum pump. After the antechamber was evacuated, it was refilled with nitrogen gas (Praxair). This procedure was repeated two more times before transferring the glassware and chemicals from the antechamber to the dry box.
3.2.2. Thin Layer Chromatography (TLC)
Thin layer chromatography was carried out on silica gel coated flexible plates
(Fluka, silica gel 60 with fluorescent indicator 254 nm) using different eluents depending
34
on the compounds involved. Specific conditions are given in 3.3. Syntheses. A drop of
sample solution was placed on the plate using a capillary and the plate was eluted with
the appropriate solvent(s). The plate was then developed dipping it in a solution of
phosphomolybdic acid in ethanol (Aldrich) and heating it with a heat gun or exposing it
to a UV lamp (λ=254 nm) or using an iodine chamber.
3.2.3. Flash Column Chromatography
Flash column chromatography was used for the purification of compounds. The
columns (Pyrex®) were first packed with silica gel as follows. The appropriate eluent
(specific conditions are given in 3.3. Syntheses) was added to a beaker containing silica
gel (Aldrich, pore size 60 Å, 0.75 cm3/g pore volume, 70-230 mesh, for column
chromatography). The resulting slurry was quickly poured into the Pyrex® column and the solvent was allowed to drain through the column until its level was just above the surface of silica gel. The column was gently tapped during this time in order to insure that there were no air bubbles in the silica gel. After the column was uniformly packed, the crude product dissolved in a small amount of solvent was carefully loaded onto the column using a pipette. The solution was allowed to drain until the level reached the top of silica gel. A small amount of sand was added carefully to protect the top of the silica column. The eluent was run through the column applying air pressure at the top of the column, resulting in rapid separation of the products and high column performance. The pressure applied was not measured but regulated by checking the speed of the solvent going out of the column that must be a fast drop by drop. The fractions were analyzed by
35
TLC and the fractions containing the pure product were combined and concentrated using a rotary evaporator under reduced pressure.
3.2.4. Nuclear Magnetic Resonance (NMR) Spectroscopy
1H and 13C NMR spectra were measured on a Brüker (400 MHz for 1H and 100
MHz for 13C) and a Varian Mercury (300 MHz for 1H and 75 MHz for 13C) spectrometer.
31P NMR spectra were measured on a Brüker spectrometer (162 MHz). Deuterated chloroform (Chemical Isotope Laboratories, 99.8% CDCl3 and Aldrich, 99.8 atom % D, contains 0.05% (v/v) TMS) or deuterated dichloromethane (Aldrich, 99.5 atom % D, contains 0.03% TMS) for the PIB samples were used as the solvents. The non-deuterated solvent trace peaks were used for internal reference: δ = 7.27 ppm (1H NMR) and δ =
13 1 13 77.23 ppm ( C NMR) for CHCl3 and δ = 5.32 ppm ( H NMR) and δ = 54.0 ppm ( C
NMR) for CH2Cl2.
3.2.5. Size Exclusion Chromatography (SEC)
Molecular weights and molecular weight distributions were determined by SEC with a Waters 150-C Plus instrument equipped with three Waters Styragel columns (HR
1, pores size = 100 Å; HR 4E, mixture of 50, 500, 1000, 10000 Å pores; HR 5E, mixture of 103, 104, 106 Å pores) thermostated at 35°C, a differential refractometer (Waters 410), and a laser light scattering detector (Wyatt Technology, DAWN EOS, λ = 690 nm). THF was employed as a mobile phase and was delivered at a flow rate of 1 mL/min. The sample solution was filtered through a PTFE 0.45µ membrane filter prior to injection in
36
the columns. ASTRA (Wyatt Technology) was used to obtain absolute molecular weight
data both with dn/dc = 0.108 for PIB in THF and using 100% mass recovery method.
3.2.6. Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS)
MALDI-TOF MS experiments were performed on a Brüker Reflex III mass spectrometer. This instrument utilizes an attenuated nitrogen laser (337 nm) with a pulse
width of approximately 3 nanoseconds. The excitation occurs over a region of 104 µm2 and irradiances are typically in the range of 106 to 107 W/cm2 (10 Hz repetition rate). The
ions were accelerated by a voltage of 20 kV. Pulsed ion extraction with 3.8 kV and 400
ns delay was applied. The instrument was externally calibrated with a polystyrene
standard (2000 g/mol) in THF (10 mg/mL). Sample solutions were prepared by mixing a solution of dithranol in THF (20 mg/mL), a solution of the polymer (10 mg/mL), and a solution of silver trifluoroacetate (AgTFA, 10 mg/mL), in a ratio of 14:4:1 (v/v/v) in the case of PIB-OH and 10:10:1 (v/v/v) in the case of PIB-O-M. The recorded spectra are the sum of 600 to 700 shots acquired at 10 Hz repetition rate.
3.3. Syntheses
3.3.1. Synthesis of 3,7-dimethyl-1-[(tetrahydro-2H-pyran-2-yl)oxy]-(Z)-2,6-octadiene (PN, product 2) - Step 1
A solution of nerol (10 g, 65 mmol) in dichloromethane (65 mL) was placed in a
250 mL one-neck round-bottom flask equipped with a condenser and a magnetic stirrer.
Dihydropyran (6.55 g, 78 mmol) and pyridinium p-toluenesulfonate (1.63 g, 6.5 mmol) were added to the reactor and the resulting solution was stirred at 27°C for 4 hours. Then
37
the reaction mixture was concentrated using a rotary evaporator under reduced pressure,
diluted with ethyl acetate and washed with saturated aqueous sodium hydrogencarbonate
solution. The organic layer was dried over magnesium sulfate, filtered and concentrated
to afford a colorless oil. The residue was purified by flash chromatography (eluent: ethyl
acetate/cyclohexane, 1:10 v/v; TLC: Rf = 0.55) on silica gel to yield PN (~14 g, 90%).
The product was analyzed by 1H and 13C NMR spectroscopies.
3.3.2. Synthesis of 2,6-dimethyl-8-[(tetrahydro-2H-pyran-2-yl)oxy]-(Z)-2,6-octadien-1-ol (PN-AOH, product 3) - Step 2
A solution of PN (13 g, 55 mmol) in dichloromethane (70 mL) was placed in a
250 mL one-neck round-bottom flask equipped with a condenser and a magnetic stirrer.
Tert-butyl hydroperoxide (24 mL, 173 mmol), selenium dioxide (0.61 g, 5.5 mmol) and salicylic acid (0.76 g, 5.5 mmol) were added to the reactor and the resulting solution was stirred at 27°C for 14 hours. Diethyl ether and water were added, the layers were separated, and the organic phase was washed with water 3 times. The combined aqueous layers were extracted with diethyl ether (4 x 30mL). The combined extracts were washed twice with saturated sodium hydrogencarbonate aqueous solution, dried over magnesium sulfate, filtered, and concentrated. The crude oil was purified by flash chromatography
(eluent: ethyl acetate/cyclohexane, 1:5 v/v; TLC: Rf = 0.1) on silica gel to yield PN-AOH
(~5 g, 35%). The product was analyzed by 1H and 13C NMR spectroscopies.
38
3.3.3. Synthesis of 1-bromo-2,6-dimethyl-8-[(tetrahydro-2H-pyran-2-yl)oxy]-(E)-2,6- octadiene (PN-ABr, product 4) – Step 3
The procedure was reported by Chen et al.181 N-bromosuccinimide (1.22 g, 6.85
mmol) dissolving in dichloromethane (10 mL) was added dimethyl sulfide (0.53 g, 8.56
mmol) under argon at -35°C and the reaction was maintained at this temperature for an
additional 1h before warmed up to 0°C. The reaction was then cooled to -35°C prior to
the introduction of PN-AOH (1.43 g, 5.62 mmol) in dichloromethane (5 mL) dropwise
followed by slowly warming up to 27°C within an hour. The reaction was worked up by
pouring the reaction mixture into saturated NaHCO3 aqueous solution. Extractions were
performed with ethyl acetate and the combined organic layers were dried over MgSO4, and concentrated. The crude product was purified by flash chromatography on silica gel to yield PN-ABr (1.64 g, 92%): TLC (ethyl acetate/hexane, 1:8, v/v) Rf 0.4.
3.3.4. Reaction of PN-ABr and 2-phenyl-1-propanol using an Alkali Metal Carbonate
A magnetic stirrer, a solution of PN-ABr (3.10 g, 9.8 mmol) in
dimethylformamide (25 mL) and potassium carbonate K2CO3 (2.2 g, 16 mmol) were
added to a 100 mL one-neck round-bottom flask. 2-phenyl-1-propanol (2.73 g, 20 mmol)
was added dropwise to the resultant solution that was stirred at 27°C for 5 h. The reaction
mixture was then poured into ice-water, extracted 3 times with ethyl acetate, dried over
magnesium sulfate, filtered, and concentrated. Tin Layer Chromatography of the crude oil (eluent: ethyl acetate/cyclohexane, 1:5 v/v) as well as 1H NMR spectroscopy were run
and showed that no reaction occured.
39
3.3.5. Reaction of 3-bromo-2-methylpropene and 2-phenyl-1-propanol using Sodium Hydride
Sodium hydride (0.511 g, 21.3 mmol) and 2-methyl-2-propen-1-ol (0.92 mL, 6.6
mmol) in solution in 30 mL of dimethylformamide were added under nitrogen and at 0°C
to a 100 mL one-neck round-bottom air-free flask equipped with a Teflon Rotaflo® stopcock, an adapter and a magnetic stirrer. The mixture was stirred at 0°C for 1h. After warming to room temperature, 3-bromo-2-methylpropene (1.0 mL, 9.9 mmol) was added dropwise and the mixture was let stirred for 45h. Then the reaction mixture was poured into water at 0°C, washed with a sodium sulfite solution (Na2SO3), and extracted 3 times
with diethyl ether. The organic layers were washed twice with water, once with a
saturated solution of sodium chloride, dried over magnesium sulfate, filtered, and
concentrated. The yield obtained was about 90%. The crude product was analyzed by 1H and 13C NMR spectroscopies.
3.3.6. Reaction of PIB-OH and 3-bromo-2-methylpropene Using Sodium Hydride
Sodium hydride (0.024 g, 1.0 mmol) and PIB-OH #16 (1.02 g, 0.18 mmol) in
solution in 15 mL of tetrahydrofuran (containing a few drops of dimethylformamide)
were added under nitrogen and at 0°C to a 100 mL one-neck round-bottom air-free flask
equipped with a Teflon Rotaflo® stopcock, an adapter and a magnetic stirrer. The mixture
was stirred at 0°C for 1 hour. After warming to room temperature, 3-bromo-2-
methylpropene (0.05 mL, 0.50 mmol) was added dropwise and the mixture was let stirred
for 27 hours. Then the reaction mixture was poured into water at 0°C, washed with a
sodium sulfite solution (Na2SO3), and extracted 3 times with diethyl ether. The organic
layers were washed twice with water, once with a saturated solution of sodium chloride, 40
dried over magnesium sulfate, filtered, and concentrated. The crude polymer was
analyzed by 1H and 13C NMR spectroscopies.
3.3.7. Synthesis of 2-methyl-2-propene p-toluenesulfonate Model Compound M-OTs Using Triethylamine
Tosyl chloride (7.48 g, 39.2 mmol) in dichloromethane (50 mL) and 2-methyl-2- propen-1-ol (1.0 mL, 11.8 mmol) were added to a 250 mL one-neck round-bottom flask equipped with a magnetic stirrer. The reaction flask was closed with a rubber stopper, put under nitrogen atmosphere and distilled triethylamine (8.8 mL, 62.8 mmol) was dropwise added to the solution using a syringe. The reaction was stirred at RT for 20h. The resulting mixture was washed 7 times with deionized water. The aqueous phase was extracted 3 times with diethyl ether. The combined organic layers were dried over magnesium sulfate, filtered, and concentrated. A TLC plate was run in an ethyl acetate/hexane (1:9 v/v) eluent.
3.3.8. Synthesis of 2-methyl-2-propene p-toluenesulfonate Model Compound M-OTs Using Sodium Hydroxide
A solution of tosyl chloride (6.30 g, 33.1 mmol) in 2-methyl-2-propen-1-ol (5.0 mL, 59.1 mmol) was added to a 100 mL three-neck round-bottom flask equipped with a dropping funnel, a reflux condenser and a magnetic stirrer. The reaction flask was immersed in an ice water bath. Sodium hydroxide solution (5N, 20 mL) was added dropwise to the mixture through the dropping funnel under a nitrogen atmosphere, following by the addition of more tosyl chloride (6.30 g, 33.1 mmol) and 5N sodium hydroxide (20 mL). The reaction mixture was then stirred for 1h. The resultant aqueous
41
solution was extracted 4 times with diethyl ether. The organic phase was washed 3 times
with a 10% sodium hydroxide solution, 3 times with deionized water, dried over
magnesium sulfate, filtered, and concentrated. The crude oil was purified by flash
chromatography (eluent: ethyl acetate/hexane, 1:9 v/v; TLC: Rf = 0.4) on silica gel to
yield M-OTs (~2.5 g, 20%). The product was analyzed by 1H and 13C NMR spectroscopies.
3.3.9. Synthesis of PIB-O-M
Dry PIB-OH (1.20 g, 0.22 mmol) was dissolved in 10 mL toluene. The solution
was then dried over magnesium sulfate, filtered, and poured into a 250 mL three-neck
round-bottom flask equipped with an adapter with stopcock and two glass caps. The solvent was removed using a vacuum pump connected to a trap. Toluene (~20 mL) distilled over benzophenone/sodium was added to the flask, the PIB-OH solution was stirred, and the solvent removed using a vacuum pump. This procedure was repeated two times, and then the PIB-OH was dried on vacuum for 1 week. THF (25 mL) distilled over benzophenone/sodium and an excess of sodium hydride (0.073 g, 3.04 mmol) were added to the flask in a dry box (nitrogen atmosphere). A magnetic stirrer and a condenser equipped with an adapter with stopcock were added to the reaction flask in the dry box, and then the mixture was heated to 80°C under nitrogen and stirred for 1h30. An excess of 2-methyl-2-propene p-toluenesulfonate (M-OTs, 0.140 g, 0.61 mmol) was added through the septum, which was then replaced with a glass cap, and the reaction mixture was stirred at 90°C for 5 days under nitrogen. The solution was filtered through celite and concentrated. The residual polymer was dissolved in ~30 mL hexane and precipitated in
42
~600 mL methanol. The obtained PIB-O-M product was dried under vacuum for 1 week and analyzed by 1H and 13C NMR spectroscopies, MALDI-TOF mass spectrometry and
SEC.
3.3.10. Bromination of 3-methyl-2-buten-1-ol
A solution of 3-methyl-2-buten-1-ol (3 mL, 29.5 mmol) in dichloromethane (100
mL) was placed in a 250 mL two-neck round-bottom air-free flask equipped with a
® Teflon Rotaflo stopcock, an adapter and a magnetic stirrer. Triphenylphosphine PPh3
(9.18 g, 35 mmol) and tetrabromomethane CBr4 (11.61 g, 35 mmol) were added to the
reactor at 0°C under nitrogen. The mixture was stirred under nitrogen atmosphere for 1h
at 0°C, then for 4h at room temperature. The solution was concentrated to afford crude
product, which was analyzed by 1H and 13C NMR spectroscopies.
3.3.11. Synthesis of Tris(tetra-n-butylammonium) Hydrogen Pyrophosphate [(NBu4)3HP2O7]
The procedure was reported by Poulter et al.182 A solution of 3.33 g (15 mmol) of disodium dihydrogen pyrophosphate Na2H2P2O7 in 41 mL of aqueous solution of
ammonium hydroxide NH4OH 0.104N (4.26 mmol) was passed through a column (L:
20cm; Ø: 1cm) of Dowex AG 50W-X8 cation-exchange resin (100-200 mesh, hydrogen
form). The free acid was eluted with 90 mL of deionized water, and the resulting solution
(pH = 1.5) was immediately titrated to pH 7.3 with 40% (w/w) aqueous tetra-n-
butylammonium hydroxide NBu4OH. The resulting solution was concentrated under vacuum and then dried by lyophilisation to yield 13g of a hygroscopic white solid (96%).
1 13 31 The product was analyzed by H, C and P NMR spectroscopies.
43
CHAPTER IV
RESULTS AND DISCUSSION
4.1. Natural Rubber Biosynthesis: A Living Carbocationic Polymerization?
The review of the biochemical literature presented in CHAPTER II led us to postulate that the biosynthesis of NR may proceed by a living carbocationic polymerization process. In this section we analyze information pertaining to the biosynthesis of NR in the biochemical literature, and translate it into polymer chemical formalism, i.e., initiation, propagation and termination. Then we propose a mechanistic view of this process.
4.1.1. The Mechanism of Natural Rubber Biosynthesis: A Living Carbocationic Polymerization?
While some of the basic steps of NR biosynthesis have been elucidated from the biochemical point of view, our understanding of this process in terms of synthetic polymer chemistry is practically nonexistent. We wish to present a new view of NR biosynthesis from the point of view of synthetic polymer chemistry. Recent insight into the mechanism and kinetics of living carbocationic polymerization183 was essential to develop this proposal.
44 In regard to polymer chemical terminology, the various allylic pyrophosphates are intitiators, the IPP is the monomer, and the rubber transferase in association with the divalent cation cofactors is the coinitiator. Thus the elementary steps of NR biosynthesis can be described in terms of initiation, propagation and termination.
4.1.1.1. Initiation
A close inspection of NR biosynthesis leads us to postulate that the structures of the intermediates involved in this process are consistent with a carbocationic polymerization mechanism. Figure 4.1 shows the proposed mechanism for geranyl pyrophosphate (GPP) initiator; however, the same process could also be formulated with neryl (NPP) pyrophosphate, etc.
45 OOOO P P O O O
GPP
OOOO P P O O O
OOOO P P O O O
IPP
OOOO P P O O O HH OOOO + P P O O O
O O O P P OOOO OOOO + P P HO O O
Figure 4.1. Proposed initiation mechanism in natural rubber biosynthesis (En = enzyme).
Initiation starts by an enzyme (and divalent cationic cofactor) – assisted ionization
of the carbon-oxygen bond of the initiator (GPP, etc.) and yields an allylic cation plus
pyrophosphate counteranion; the enzyme plus cofactor(s) coordinates with the pyrophosphate “protecting” group and mediates the formation of the initiating 46 carbocation. According to polymer chemical convention, the enzyme plus cofactors constitute the coinitiating system (Figure 4.2).
En
O O O O 2+Mt P P Mt2+ O O O Mt2+ = Mg2+ or Mn2+ En = enzyme
En O O O O 2+Mt P P Mt2+ O O O
Figure 4.2. Possible role of divalent cations in natural rubber biosynthesis.
Ionization at the chain end is favored by resonance stabilization of the allylic carbocation and increasing entropy of the system. This mechanism is unlikely with the
IPP monomer because the cleavage of the carbon-oxygen bond would lead to an energetically unfavorable primary carbocation. Subsequently, the vinylidene group of
IPP adds to the allylic carbocation, yielding a tertiary carbocation which, via proton elimination, regenerates the trisubstituted allylic pyrophosphate. This mechanism applies to the formation of trans-1,1-dimethylallylic initiators (see Figure 2.11), catalyzed by trans-prenyl transferase, as well as to the incorporation of the first cis-unit, i.e., initiation, catalyzed by cis-prenyl transferase (Figure 4.1). In regard to trans or cis stereoregulation, the specific enzyme functions as the template, and yields exclusively trans-1,4 or cis-1,4
47 incorporation. The incorporation of each IPP unit is always accompanied by the loss of
pyrophosphoric acid HPP (or its salts).
4.1.1.2. Propagation
Propagation proceeds by the same cationation/IPP-addition/proton loss sequence as occurs in initiation (Figure 4.1). The initiation and propagation steps are fundamentally similar to those in living carbocationic (and living radical) polymerization governed by dormant-active equilibria.184,185 The difference is that in the natural process every
propagation step is followed by deactivation, accompanied by the release of HPP, while
in living carbocationic (or radical) polymerization the active chain end adds more than
one monomer units before temporary deactivation.183,184 Poulter et al.’s work on prenyl
transfer reactions in natural terpenoid synthesis supports the possible involvement of
carbocationic species in NR biosynthesis (see CHAPTER II).138,166-168
A unique feature of in vivo NR biosynthesis is the control of proton loss from the tertiary carbocation yielding exclusively 1,4-enchainment (III in Figure 4.3). According to classical organic chemistry proton loss from a tertiary carbocation leads to isomer mixtures I, II and III (Figure 4.3).
48 H3C CH3
CCHCH2 CH2 C CH2 CH2 PP
H3C R - H
CH3 CH3
R CH C CH2 CH2 PP R CH2 C CH CH2 PP (cis and trans)(cis and trans) IIII
CH2
R CH2 C CH2 CH2 PP
II
Figure 4.3. Isomers obtained by proton loss from a tertiary carbocation.
The exclusive formation of III in vivo suggests a more acidic proton between the tertiary
carbocation and the –CH2-PP group, or a concerted mechanism involving one of the
pyrophosphate groups and/or the enzyme. Hevea rubber biosynthesis in vivo also yields
exclusively cisIII, but as mentioned above, some plants produce transIII, or a mixture of
cis and transIII.
The proposed carbocationic mechanism is consistent with the observed broad
molecular weight distributions and cyclized sequences present in NR; intermolecular
attack of the growing carbocation on a double bond of another polymer chain may lead to
49 branching and broad/multimodal distributions, and intramolecular attack to cyclization.
Fresh latex was shown to exhibit monomodal molecular weight distribution60,61, while processed Hevea rubber displays multimodal distribution. These facts can be readily explained by chain-chain coupling under acidic conditions. Chain-chain coupling and cyclization have been observed in the carbocationic polymerization of isoprene.173,186
Terpenoid cyclization occurring in plants is also believed to proceed by carbocationic mechanism.187 It was suggested that the “abnormal” groups arise during processing.60,61
In our view, only initiation, propagation and temporary deactivation proceed in vivo. Cis- trans stereoregulation may be due to specific enzymes acting as templates for monomer incorporation; Figure 4.4 helps to visualize this process.
Aqueous Enzyme medium Monolayer
Monomer
Non-polar rubber phase Phospholipid fatty acid
Figure 4.4. Visualization of the natural rubber synthesis in vivo.
Evidence for this view came from an analysis of NR biosynthesis, combined with an evaluation of recent progress in sequencing and 3D structure determination of various
50 prenyltransferases enzymes,187 as well as other biochemical studies concerning the mechanisms of isoprenoid syntheses.187 For example, in prenyl transfer reactions the binding sites for the initiating 1,1-dimethylallylic pyrophosphates and IPP were located within the hydrophilic regions of the enzyme, whereas chain growth was proposed to take place within a hydrophobic pocket positioned toward the bottom end of the conical enzyme (Figure 4.5).
Hydrophilic DDxxD(1) (2)DDxxD pocket PP PP
Hydrophobic pocket
Figure 4.5. A scheme of the active sites in avian trans-prenyltransferase.187 DDxxD –Aspartic acid rich motifs; the oval represents a large amino acid proposed to stop chain growth.
According to our view, the amphiphilic enzyme resides at the interface (Figure
4.4). Propagation occurs as suggested,187 when the allylic end of the initiator or the NR molecule is docked at the specific site within the tunnel-like crevice of the enzyme and is activated, and IPP assisted by divalent cofactors (not shown in Figure 4.4) enters from the aqueous phase. Stereoregulation within the interior of the enzyme is visualized by steric restriction – the spatial arrangement and size of the various microstructures shown in
51 Figure 2.3 are compatible with a specific enzyme pocket. The rubber molecule may be
released from the docking site after the incorporation of IPP, and a new rubber molecule
may be docked and activated. The rubber molecules compete for the active sites; this
alone can lean to broad molecular weight distributions. Continuous supply of IPP
monomer and APP initiator to the particle will also lead to broad distribution. Chain
growth stops when the rubber chain reaches a critical molecular weight, which prevents
further diffusion of large molecules to the interface. The critical molecular weight is
species-dependent, and is most likely based on the stabilization and composition of the
latex. Permanent termination does not occur in vivo, only during processing.
4.1.1.3. Termination
Termination in isoprenoid biosynthesis was proposed to be due to the position of
specific large motifs (see Figure 4.5) that stop chain growth at a specific length.187 This is an attractive suggestion for the formation of short isoprenoids, but not for Hevea rubber that has ~ 5000 repeat units. With increasing chain length the NR molecule becomes increasingly hydrophobic and will extend beyond the enzyme pocket; thus chain length regulation by this mechanism is inconceivable. According to the biochemical literature pertinent to NR biosynthesis, chain termination occurs when the enzyme relinquishes the rubber molecule. If the detachment of the enzyme (termination) were to occur at a specific chain length regulated by steric factors as suggested for short-chain isoprenoids, uniform NR would arise. Indeed, such short-chain isoprenoids with well-defined chain lengths (up to 24-mers) arise in plants catalyzed by various cis- and trans- prenyltransferases.187 However, NR exhibits broad molecular weight distribution. The
52 proposed chain activation involving the enzyme and cofactors (see Figure 4.1) implies
that the enzyme is released after each propagation step. Evidently, the polymerization rate
is governed by the rate of IPP addition relative to that of activation of the dormant rubber
molecule. Random activation and migration/docking of the rubber molecule to the
activating site of the enzyme (see Figure 4.4) lead to broad molecular weight
distributions.
The NR molecule contains HO-end groups, which most likely arise by hydrolytic
cleavage of terminal polymer-pyrophosphate linkages (Figure 4.6).
PP
0 to 3 n + 1
+ H /H2O OH + HPP 0 to 3 n + 1
Figure 4.6. Termination: hydrolysis of the chain end.
The chain end may also react with fatty acids, which leads to ester end groups. It is unknown whether hydrolytic termination occurs in vivo or during processing. At physiological pH, the pyrophosphate end group is a quite stable dianion.167 Post- polymerization reactions have been described.60,61 We believe that termination by
hydrolysis does not occur in vivo, because this would prevent the formation of high
53 molecular weight rubber. Species-specific molecular weight regulation is conceivably due to physical factors, such as the size of the latex particle and/or stabilization by specific proteins in the plant.
4.1.2. Conclusion: Natural Living Carbocationic Polymerization (NLCP)
Based on the above comprehensive review and analysis of the chemical, polymer chemical and biochemical literature pertaining to the biosynthesis of NR, we developed a new mechanistic view of this process. First, we wish to stress that this biosynthesis proceeds by a chain growth (addition) mechanism that fits the definition of living polymerization set forth by the IUPAC: “Living polymerization is a chain polymerization from which chain transfer and chain termination are absent.” Second, all the species identified by earlier authors that are known to arise during biosynthesis can be described in terms of carbocationic intermediates. A combination of these two critical parameters, livingness and carbocationic intermediates, leads us to propose that the biosynthesis of
NR proceeds by a natural living carbocationic polymerization mechanism (NLCP).
Figure 4.7 summarizes the concept of NLCP.
54 Initiation
X X Ionization Y (priming) Initiator
X X Cationation X + X
Monomer (MX)
HX
X
Propagation
X Y X
n n
X HX X X X Y +
MX n n+1 n+1
Figure 4.7. Proposed natural living carbocationic polymerization mechanism.
The polymerization starts by initiation, which involves two events: ionization or priming
(tantamount to activation in biochemical parlance) and cationation. During ionization by an activator (Y) the allylic end group of an initiator yields a resonance stabilized primary/tertiary allylic cation. In NR biosynthesis ionization is mediated by specific transferases and assisted by inorganic cofactors (see Figure 4.2), while the NLCP concept
55 is inclusive of other possible activators that produce the desired resonance-stabilized
allylic cation. If the activated chain end cationates incoming monomer (MX), a tertiary
carbocation intermediate (shown in brackets) is formed which promptly loses a proton
(i.e., HX is lost). MX is a “protected” monomer that cannot be ionized by the activator Y.
The counteranion X- is a pyrophosphate residue in plants; however in NLCP it may be a
different anion. Propagation is repetitive ionization/cationation: the allylic terminus is
ionized (activated) by Y, reacts with the incoming monomer, and the intermediate tertiary
carbocation, via HX loss, regenerates exclusively the allylic chain end. This sequence of
events sustains propagation by the same mechanism that prevails in initiation. Thus
NLCP can be viewed as a controlled/living carbocationic polymerization with dynamic equilibria between dormant and active species, combined with controlled loss of HX.
Importantly, HX loss after each monomer incorporation must regenerate exclusively the dormant allylic chain end - III in Figure 4.3.
+ - Pn – X + Y Pn // X-Y
+ - + - Pn // X-Y + MX → Pn+1 X // X-Y → Pn+1 – X + HX + Y
The NLCP mechanism, combined with cis-trans stereoregulation (for example, by
the activator or by a suitable template), may serve as a blueprint for the design of a
synthetic system emulating the biosynthesis of NR.
56 4.2. Synthesis of Model Intermediates for the Preparation of Polyisobutylene-Neryl Pyrophosphate (PIB-NPP) Macroinitiator
This part of the project was also inspired from the review of the biochemical literature presented in CHAPTER II. Indeed the ultimate purpose is to verify if the cis- prenyltransferase enzyme would recognize a new synthetic molecule as an initiator of cis-
1,4-polyisoprene in vitro biosynthesis, and if successful, to produce PIB-b-NR diblock copolymer. Thus the plan consisted of the synthesis of a polyisobutylene chain carrying a neryl pyrophosphate functional group (PIB-NPP), from PIB-OH made by “traditional” living carbocationic polymerization.
The strategy developed for the synthesis of PIB-NPP was inspired by Chen et al.’s work181 and is outlined in Figure 4.8.
57 tBuOOH, SeO OH PPTs 2 DHP salicylic acid
CH2Cl2 CH2Cl2 OH 27°C, 4h OO27°C, 14h OO 1 step 1 2 step 2 3 Nerol N PN PN-AOH
NBS Br K2CO3 OPIB (CH3)2S PIB-OH
CH2Cl2 DMF -35°C to 27°C OO27°C, 4h OO 3h step 4 step 3 4 5 PN-ABr PIB-PN
PPTs OPIBa) CBr4, PPh3, OPIB EtOH CH2Cl2, 27°C, 4h O O
55°C, 8h b) (n-Bu4N)3HP2O7, OH CH CN, 27°C, 6h OPOPO step 5 3 6 step 6 7 O O PIB-N PIB-NPP
with PIB-O =
CH3 CH3 CH3
C CH2 C CH2 C Cl n CH2 CH3 CH3 O with PPTs = pyridinium p-toluenesulfonate; DHP = dihydropyran; NBS = N-bromosuccinimide
Figure 4.8. Synthetic strategy to produce the PIB-NPP macroinitiator.
In step 1, the hydroxyl group of the commercially available starting compound, nerol (N, product 1), was protected with tetrahydropyranyl ether. Next the protected nerol
(PN, product 2) was oxidized (step 2) to the corresponding allylic alcohol (PN-AOH,
58 product 3). Subsequently the hydroxyl group was replaced with Br (step 3) to yield an allylic bromide (PN-ABr, product 4). In step 4, PIB-OH (PIB carrying a primary
hydroxyl head group) was used to obtain compound 5 (PIB-PN), which was then
deprotected (step 5, PIB-N, product 6) and functionalized (step 6) to have a
pyrophosphate end group (PIB-NPP, product 7). PIB-NPP was our ultimate target
product.
The 1H NMR spectrum of nerol N is shown in Figure 4.9.
eg010 (g) Brüker 400MHz 1 (h) (c) Nerol, H NMR CH3 (g) (e) (d) CH2 (h) CH CH CH 2 (f) 3 (d)(e) CH2 (a) H3C CH OH (c) (b) (i)
(a) cyclohexane
(i) ? (b) (f)
Integral 0.9558 0.9673 2.0000 4.0949 0.7078 3.1509 3.1390 3.1566 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 (ppm)
Figure 4.9. 1H NMR spectrum of nerol (N, product 1).
The two resonances (b) and (f) in the 5 to 5.5 ppm region of the spectrum
correspond to the methyne protons: 5.05-5.12 (m, 1H), 5.43 (t, 1H, J = 6.9Hz); “m”
59 stands for multiplet and “t” for triplet. The multiplets arise from spin-spin couplings that
are transmitted through chemical bonds and yield information about the immediate
molecular environment. In the case of the proton (b) signal, it is split by the two
neighboring protons of the CH2 (a) to yield a triplet pattern. J refers to the coupling
constant of the triplet. The case of the proton (f) is slightly different; indeed it is
perturbed by a set of nearby spins so that it manifests its response to such influence in its
resonance signal by showing up as a multiplet. The same is true in all other cases where the proton resonance signal appears to be a multiplet. The hydrogens of the CH2 group
(a) next to the oxygen show up as a doublet at 4.07 ppm (d, 2H), since it is influenced by the methyne proton (b). The four protons of the two CH2 groups, (d) and (e), are found at
2.02-2.13 (m, 4H). Finally the CH3 groups (c), (g), (h) are identified as singlets at 1.59,
1.67 and 1.72 ppm respectively.
4.2.1. Syntheses of the Protected Nerol PN (step 1) and the Allylic Alcohol PN-AOH (step 2)
Steps 1 and 2 were carried out in the “Laboratoire de Chimie des Polymères
Organiques” (LCPO), Pessac, France. Chen et al.181 described experimental conditions for geranol. We used commercially available nerol as a starting material and carried out the experiments as described in CHAPTER III, 3.3.1 and 3.3.2. The procedure for the purification of the crude products was modified. At LCPO we were not allowed to use hexane for flash chromatography column because of toxicity concerns. Thus we replaced hexane with cyclohexane, whose polarity is close to that of hexane (polarity indexes: hexane = 0 and cyclohexane = 0.2).188 Thin Layer Chromatography was run in both cases
60 to determine the best eluent needed for flash chromatography. In the case of step 1, a 1:10 v/v mixture of ethyl acetate and cyclohexane provided the best separation between the pure PN and impurities and led to a retention factor of Rf = 0.55 for the pure compound.
In the case of step 2, a 1:5 v/v mixture of ethyl acetate an cyclohexane was used and gave a Rf = 0.1 for the pure PN-AOH. Pure compounds with yields in good agreement with the literature (90-95% for PN and 35-40% for PN-AOH) were obtained. The 1H and 13C
NMR spectra of PN and PN-AOH are shown in Figures 4.10, 4.11, 4.12 and 4.13. The first trial of each step was not convincing (55% yield for step 1 and 0% yield for step 2) but these experiments were then carried out 3 times for each step providing each time the results claimed above. Brüker 400 MHz cyclohexane
(g) CH 3 (c) (f) (h) HC CH3 (g) (P3) H2 CH C (d) 2 (P2) (P4) H2C (e) H2C CH2 (h) (a) H2 C CH CH2 (c) H3COC (P1) O (P5) H (b) (d)(e) (P2)(P3) (P1) (P5) (P4) (b) (f) (a)
Figure 4.10. 1H NMR spectrum of 3,7-dimethyl-1-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadiene (PN, product 2).
61 Figure 4.10 is the 1H spectrum of the pure compound. The desired protected nerol
(PN) can be identified. The two resonances (b) and (f) corresponding to the methyne protons are found in the 5 to 5.5 ppm region of the spectrum: 5.05-5.12 (m, 1H), 5.35 (t,
1H, J = 6.9 Hz). (P1) at 4.59-4.63 (m, 1H) is assigned to the proton on the carbon in the tetrahydropyranyl ring that is attached to the oxygen of the protected nerol. The hydrogens of the two CH2 groups (a) and (P5) next to the oxygen of the PN are not equivalent and show up as four multiplets at 3.45-3.51 (m, 1H), 3.84-3.90 (m, 1H), 3.93-
4.00 (m, 1H), 4.15-4.22 (m, 1H). The four protons of the two CH2 groups of the nerol, (d) and (e), are found at 2.00-2.10 (m, 4H). The CH2 protons of the protecting tetrahydropyranyl ring, (P2), (P3) and (P4), appear as a multiplet in the 1.4 to 1.8 ppm region. Finally the CH3 groups (c), (g), (h) are identified as singlets at 1.58, 1.65 and 1.72 ppm, respectively. Integration of the peaks corresponds exactly to the ratios expected from the chemical formula of PN, demonstrating that pure product was obtained.
62 Brüker 400 MHz 9
7 6 8 C 5 4 B D
1 3 E A 10 O O 2 5 10
B A E 1 4 8 C 6 D 2 9
3 7
Figure 4.11. 13C NMR spectrum of 3,7-dimethyl-1-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadiene (PN, product 2).
13C NMR supports this conclusion. In Figure 4.11, all expected carbon resonances can also be identified. The four carbons (2), (3), (6), (7) of the two double bonds appear
in the 120-140 ppm region. The resonance at 98 ppm corresponds to the carbon (A)
between the oxygen of the protected nerol and the oxygen in the tetrahydropyranyl ring.
The resonances at 62 and 63 ppm correspond to the carbon atom next to the oxygen in the
protected nerol moiety (1), and the carbon next to the oxygen in the tetrahydropyranyl
ring (E), respectively. Finally the group of resonances starting at 18 ppm and ending at
32 ppm includes the CH2 carbons of nerol (4) and (5), the protective ring (B), (C), (D), and the methyl carbons of the protected nerol (8), (9) and (10).
63
Brüker 400 MHz (c) (g) (g) CH3 (i) (f) OH HC C (h) H2 (P3) (h) H2 (d) CH2 (P2) C H2C (e) H2C CH2 (P4) (a) H2 (d) (e) C CH CH2 (P5) H3COC (P1) O (c) H (b) (P2)(P3) (P1) (P4) (b) (f) (a) (P5) (i)
Figure 4.12. 1H NMR spectrum of 2,6-dimethyl-8-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadien-1-ol (PN-AOH, product 3).
Figure 4.12 shows that the oxidation of PN to yield PN-AOH was successful.
Indeed, a new resonance (i) appeared at 2.5 ppm, which can be assigned to the proton of
the allylic alcohol. The signal (h) at 3.9 ppm is assigned to the CH2 protons next to the allylic OH. Integration of (i) and (h) yielded the expected ratio of 1:2.
64
Brüker 400 MHz 9
7 OH 6 8 C 5 4 B D
3 1 E 10 O A O 2 D
8 6 2 A E B 5 10 4 C 1 9 7 3
Figure 4.13. 13C NMR spectrum of 2,6-dimethyl-8-[(tetrahydro-2H-pyran-2-yl)oxy]- (Z)-2,6-octadien-1-ol (PN-AOH, product 3).
The 13C NMR shown in Figure 4.13 verifies our conclusion: (8) shifted from 24 to
68 ppm, corresponding to the CH2 carbon next to the oxygen of the OH group. Once again, it can be concluded that the oxidation of PN into an allylic alcohol was successful.
65 4.2.2. Synthesis of the Allylic Bromide PN-ABr (step 3)
Step 3 was carried out following the experimental procedure described in literature by Chen et al. and reported in CHAPTER III, 3.3.3.181 The two first trials led to traces of pure PN-ABr. We were not able to isolate it. The third experiment led to the pure desired compound in only 8% yield. The purification method is described below.
Being unsuccessful reproducing the experimental procedure described in the literature (see CHAPTER III, 3.3.3.), we contacted the authors. Chao-Tsen Chen from
Department of Chemistry of National Taiwan University answered providing new information. Here is thus the detailed successful procedure carried out. The details previously missing are highlighted in italic.
A solution of N-bromosuccinimide (2.56 g, 14.38 mmol) in dichloromethane (20 mL) was placed in a 100 mL one-neck round-bottom air-free flask equipped with a
Teflon Rotaflo® stopcock, an adapter and a magnetic stirrer. Dimethyl sulfide (1.12 g,
17.98 mmol) was added under nitrogen at –35°C. The reaction flask was kept in an
ethanol bath that had been cooled by pouring liquid nitrogen into it while stirring; the
temperature was continuously checked with a thermometer. The reaction mixture was
maintained at this temperature (-35°C) for an additional hour and was then warmed up to
0°C (the reaction flask was transferred into an ice water bath) and stirred for 1 more hour
at that temperature. The reaction was then cooled again to –35°C prior to the dropwise
introduction of PN-AOH (3.0 g, 11.8 mmol) in dichloromethane (10 mL) followed by
slowly warming up the mixture to 27°C within an hour. The mixture was stirred at that
temperature until N-bromosuccinimide was completely dissolved in the solution. The
66 reaction was worked up by pouring the reaction mixture into saturated sodium hydrogencarbonate aqueous solution. The aqueous phase was extracted with ethyl acetate
(5 x 5mL) and the combined organic layers were dried over magnesium sulfate, filtered, and concentrated using a rotary evaporator under reduced pressure. The crude product was finally purified by flash chromatography (eluent: ethyl acetate/cyclohexane, 1:8 v/v;
TLC: Rf = 0.6) on silica gel to yield pure PN-ABr in 45% yield. The pure product was analyzed by 1H and 13C NMR spectroscopies. The spectra are shown in Figures 4.14 and
4.15.
Brüker 400 MHz cyclohexane
(g) CH3 (P2)(P3) (f) Br (P4) HC C (h) H2 (P3) (g) (c) H2 Silicone (d) CH2 (P2) C grease H2C (e) H2C CH2 (P4) (a) H2 (h) C CH CH2 (P5) H3COC (P1) O (c) H(b)
grease (d) (e) (P1) (P5) (f)(b) (a)
Figure 4.14. 1H NMR spectrum of 1-bromo-2,6-dimethyl-8-[(tetrahydro-2H-pyran- 2-yl)oxy]-(Z)-2,6-octadiene (product 4).
67 PN-ABr can be identified from the 1H NMR spectrum (Figure 4.14) even if it is not very different from the spectrum of PN-AOH in Figure 4.12. The peak (h) assigned to
the CH2OH protons is not shifted much. However the broad singlet (i) assigned to the
proton of the allylic alcohol is absent.
Brüker 400 MHz 9
7 Br 6 8 C 4 5 B D
3 1 E 10 O A O 2
cyclohexane grease
Silicone 6 A E1 8 45D C grease 7 3 2 B 10 9
Figure 4.15. 13C NMR spectrum of 1-bromo-2,6-dimethyl-8-[(tetrahydro-2H-pyran- 2-yl)oxy]-(Z)-2,6-octadiene (product 4).
13 In Figure 4.15 the C NMR spectrum presented shows that the CH2 peak (8) was
shifted from 68 to 43 ppm. This observation gives the evidence that pure PN-ABr was
obtained after the flash chromatography column.
68 The yield obtained did not really agree well with literature; indeed the authors
claim a 90% yield.181 Trials were made to increase the yield of this reaction. PN-AOH
was replaced by a model compound for these experiments. The model compound used is
the 2-methyl-2-propen-1-ol (Figure 4.16).
OH
Figure 4.16. Structure of 2-methyl-2-propen-1-ol.
Two trials were made using the same conditions described in CHAPTER III, but
remained unsuccessful. Indeed, NMR spectra of crude product were not convincing, and
a too small amount of crude product was obtained to be purified by flash chromatography
since only 1 mL of alcohol was used. A third trial was made also using the same
conditions to make sure that they worked for the model compound; a larger amount of
alcohol was used (3 mL). From the 1H NMR spectrum (Figure 4.17) that 3-bromo-2-
methylpropene was synthesized (see corresponding (a), (b), (c), (d) singlets on the
spectrum) but nothing can be concluded with certainty from the 13C NMR spectrum
(Figure 4.18) since no carbon peak from the desired molecule could be identified.
Unfortunately nothing can be known about the yield since time was missing to carry out a column chromatography.
69
1H eg.111 - CDCl3 - 27/07/2005brominated model compoud NBS Me2S
eg.111 - CDCl3 - 27/07/2005brominated model compoud NBS Me2S a CH3 c Br H C H 2 b H d a b
d c 1.0000 1.0043 2.0429 3.1752 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 (ppm)
10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 (ppm)
Figure 4.17. 1H NMR spectrum of the brominated model alcohol.
70
eg.111 - CDCl3 - 27/07/2005brominated model compoud NBS Me2S
190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 (ppm)
Figure 4.18. 13C NMR spectrum of the brominated model alcohol.
71 4.2.3. Model Reactions for the Synthesis of PIB-PN (product 5)
Several attempts were made to find a way to link the protected nerol PN to the
PIB macromolecule. Model compounds were mostly used for that purpose. The results of
these experiments are detailed in the following part.
4.2.3.1. Reaction of PN-ABr and an Alcohol Model Compound Using Alkali Metal Carbonate
In this set of experiments, PN-ABr was reacted with 2-phenyl-1-propanol (Figure
4.19), a model compound, which is the molecule commercially available that mimicks
the best the end of the PIB-OH macromolecule.
OH
Figure 4.19. Structure of 2-phenyl-1-propanol.
Chen et al.181 obtained an ether molecule by reacting a compound similar to PN-
ABr (the same but starting from geranol instead of nerol) and 7-hydroxy-8-methyl-4- trifluoromethyl coumarin. They used anhydrous K2CO3 in dimethylformamide DMF and
ran the reaction at 27°C for 4h. We followed exactly the same procedure for the reaction
of PN-ABr and 2-phenyl-1-propanol (see CHAPTER III, 3.3.4); however the desired
ether was not synthesized.
72 Two additional trials were made; K2CO3 was replaced with Cs2CO3, DMF was dried over magnesium sulfate before use, and the reaction mixture was heated overnight at 80°C under nitrogen atmosphere. No reaction occurred. This was probably due to the low acidity of the primary alcohol used (2-phenyl-1-propanol). Thus we thought about using a stronger base to try to overcome this problem.
4.2.3.2. Reaction of a Brominated and a Hydroxyl Model Compound Using Sodium Hydride
We carried out new experiments using sodium hydride as a very strong base supposed to remove more easily the hydroxyl proton of 2-phenyl-1-propanol. We only used model compounds in the following trials in order to save the PN-ABr synthesized.
While we were waiting for the ordered 2-phenyl-1-propanol to arrive, a first experiment was made using 2-methyl-2-propen-1-ol (Figure 4.16) and 3-bromo-2- methylpropene (Figure 4.20).
Br
Figure 4.20. Structure of 3-bromo-2-methylpropene.
The desired ether was obtained in 90% yield. The experimental conditions were the same as those we mention below.
73 Then two model compounds mimicking the end of the molecules involved in the synthetic strategy (PIB-OH and PN-ABr) were reacted to form the corresponding ether.
These model molecules were 3-bromo-2-methylpropene (Figure 4.20) and 2-phenyl-1- propanol (Figure 4.19). The conditions used for this synthesis are detailed in CHAPTER
III, 3.3.5. The 1H and 13C NMR spectra are shown in Figures 4.21 and 4.22, respectively.
These spectra show that the product obtained is quite pure. However flash chromatography would be needed to provide a totally pure compound.
74 1H NMR eg.103 - CDCl3 - 09/06/2005crude ether (model compounds) 1a 1b 12 H 3 H H 9 3 H3C 2 C CH2 CH O H 11
4 4 CH 10 3 10 HC CH
5 HC CH 5 CH 6 9 4, 5, 6
1a, 1b 11, 12
2
Integral 5.8971 2.3760 2.2596 0.2981 2.3608 1.1396 0.8206 3.0000 6.1219 1.1894 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 (ppm)
Figure 4.21. 1H NMR spectrum of the ether model compound.
The desired ether can be identified. The multiplet involving the peaks 4, 5, 6 corresponding to the five aromatic protons of the phenyl ring is found in the 7.2 to 7.4 ppm region of the spectrum. Resonances 11 and 12 at 4.88 (s, 1H) and 4.92 (s, 1H) are assigned to the vinyl protons of the brominated molecule. The hydrogens of the two CH2 groups 9 and 1 next to the oxygen atom of the molecule respectively show up at 3.9 ppm
(s, 2H), and 3.53 and 3.45 ppm (dq, 2H, J1a1b = 9.1 Hz, J1a2 = 6.2 Hz, J1b2 = 7.6 Hz). The proton 2 of the CH group of the alcohol is found at 2.9-3.1 (m, 1H). Finally the CH3 groups 10 and 3 are identified respectively as singlets at 1.7 ppm (s, 3H) and 1.35 ppm (s,
3H). Integration of the peaks seems to correspond to the ratios expected from the chemical formula of the desired ether compound.
75
13C NM R eg.103 - CDCl3 - 09/06/2005crude ether (model compounds)
144.5990 142.5333 128.4439 127.4692 126.4218 112.0997 75.9635 75.0325 73.9050 68.8425 66.0129 42.5622 40.1619 32.0662 29.8476 22.8430 19.5189 18.4787 17.7150 15.4237 14.2890
110 5 32 11 13 O 6 4 5 5 12
6 6 7
2 13 1 3 7 10 12
11 4 diethyl ether diethyl ether DMF
140 130 120 110 100 90 80 70 60 50 40 30 20 (ppm)
Figure 4.22. 13C NMR spectrum of the ether model compound.
13C NMR supports this conclusion. In Figure 4.22, all expected carbon resonances can also be identified. The six aromatic carbons 4, 5, 6, 7 and the two vinyl carbons 11, 13 appear in the 110-150 ppm region. The resonances at 76 and 75 ppm correspond to the carbon atoms 10 and 1 next to the oxygen atom in the ether molecule. The carbon 2 of the
CH group of the alcohol is found at 40 ppm. Finally the peaks at 18 and 20 ppm are assigned to the methyl carbons 3 and 12.
The previous reaction was conducted in the same conditions using THF as the solvent. Similar results (amount of crude product, NMR spectra) were obtained.
76 4.2.3.3. Reaction of PIB-OH and a Brominated Model Compound Using Sodium Hydride
The same experiment as the previous ones was carried out with PIB-OH #16 (see
CHAPTER III, 3.1 for details about the sample) and 3-bromo-2-methylpropene (see detailed procedure in CHAPTER III, 3.3.6). Tetrahydrofuran was used as the reaction solvent. The 1H NMR spectrum is provided in APPENDIX C. This spectrum was carefully analyzed and compared ppm by ppm with the PIB-OH one. No convincing proof of the linkage of the new end chain was found even by comparing integration peaks: the presence of the double bond of the methylpropene was not detected on the spectra. The etherification reaction did not occur.
4.2.3.4. Tosylation of PN-AOH
As discussed, the trials previously carried out to link PIB-OH to model compounds remained unsuccessful. We then realized that it might be easier and more efficient to synthesize PIB-PN (product 5) starting from PN-AOH (product 3) via a tosylated intermediate PN-AOTs. We proposed to carry out model experiments using 2- methyl-2-propen-1-ol (Figure 4.16, M-OH) as a model compound for PN-AOH. Ideally we should have used 2-methyl-2-buten-1-ol, but this product is not commercially available. The synthetic pathway proposed is represented in Figure 4.23.
tosyl chloride PIB-OH OH O O PIB S O O M-OHM-OTs PIB-O-M
Figure 4.23. Pathway for the synthesis of PIB-PN with model compounds involving a tosylate intermediate. 77 We first adapted Aubrecht and Grubbs procedure189 to our system for the tosylation of M-OH. The procedure we followed is detailed in CHAPTER III, 3.3.7. The
TLC run showed that the yield of the reaction was very low. We did not purify the desired tosylate by column chromatography since we would have collected a too small amount to run the next step.
The same procedure was then used for a second trial. The reaction mixture was stirred at RT for 6 days and the product obtained was analyzed by TLC and by 1H NMR spectroscopy. The TLC plate showed that the yield of the reaction was still very poor.
Another procedure was then suggested (see CHAPTER III, 3.3.8). It yielded the desired model tosylate M-OTs in 20% yield after purification by flash chromatography.
The 1H and 13C NMR spectra of the pure tosylate are shown in Figures 4.24 and 4.25.
78
083106_OTscheck_CDCl3_1H.esp 7 6 H 7 5 3 H CH3 CH 3 2 H C O C CH2 S H 6 4 3 H O O H 1 5 4
5 6 21 TMS CHLOROFORM-d
1.66 1.83 1.98 2.00 3.55 3.04
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 Chemical Shift (ppm)
Figure 4.24. 1H NMR spectrum of 2-methyl-2-propene p-toluenesulfonate M-OTs.
The resonances 5 and 6 corresponding to the aromatic protons are found in the 7 to 8 ppm region of the spectrum: 7.80 (d, 2H, J = 9 Hz) and 7.35 (d, 2H, J = 9 Hz). The resonances
1 and 2 at 4.99 (s, 1H) and 4.94 (s, 1H) are assigned to the vinyl protons. The hydrogens of the CH2 group 4 next to the oxygen atom of the molecule show up at 4.43 ppm (s, 2H).
Finally the methyl groups 7 and 3 are identified respectively as singlets at 2.49 ppm (s,
3H) and 1.69 ppm (s, 3H). Integration of the peaks corresponds to the ratios expected from the chemical formula of the 2-methyl-2-propene p-toluenesulfonate model compound.
79 110106_OTs_CDCl3_13C.esp 6
9 7 3 CH3 6 8 CH3 7 2 C O 7 5 H2C CH2 S 6 1 4 O O
1
4 9 3
8 2 5 CHLOROFORM-d
145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 Chemical Shift (ppm)
Figure 4.25. 13C NMR spectrum of 2-methyl-2-propene p-toluenesulfonate M-OTs.
The 13C NMR spectrum supports this conclusion. In Figure 4.25, all expected carbon resonances can also be identified. The six aromatic carbons 5, 6, 7, 8 of the molecule appear at 133, 128, 130 and 145 ppm respectively. The resonance at 138 ppm corresponds to the carbon atom 2. The CH2 group 4 next to the oxygen atom appears at
74 ppm. The carbon 1 of the vinyl group is found at 116 ppm. Finally the peaks at 19 and
22 ppm are assigned to the methyl carbons 3 and 9.
4.2.3.5. Synthesis of PIB-O-M
The 1H NMR spectrum of the starting PIB-OH #15 is shown in Figure 4.26.
80
093006_PIBOH#15_CDCl3_1H.esp
15 16 15 87 651 3 4 16 CH CH CH CH CH CH CH HC CH 3 3 3 3 3 3 3 13 12 11 2 10 9
17 HC C C CH2 C CH2 C CH2 C CH2 C CH2 C CH2 C Cl
HC CH 14 CH2 CH3 CH3 CH3 CH3 CH3 CH3 16 15 m 7651 34 OH (b1)(b2) CH2 Impurity (a) PIB CH2 C
CH3 14 14 17 (c)
(b1)(b2)
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 Chemical Shift (ppm)
093006_PIBOH#15_CDCl3_1H.esp 1 2
3 5
grease (b )(b ) 1 2 4 CH2 8 (a) PIB CH2 C 6 CH3 (c) 10 12 11 7
9 (c) 13 (a)
2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 Chemical Shift (ppm)
Figure 4.26. 1H NMR spectrum of PIB-OH.
81 Assignments were based on previously published data.178,190 The aromatic protons of the initiating moiety are found in the 7.20-7.40 ppm region of the spectrum: 15 (m,
2H), 16 (m, 2H) and 17 (m, 1H). The CH2 protons 14 adjacent to the hydroxyl group show up as two doublets at 3.68 (1H, J = 10.5 Hz) and 3.44 ppm (1H, J = 10.5 Hz). These doublets represent diastereometric protons that are adjacent to an asymmetric center and, therefore, the splitting of each other’s signal with geminal two-bond coupling (2J). The proton signal of the hydroxyl head group, expected at 2.84 ppm, can not be seen because of its fast exchange with the CH2 protons adjacent to it. The resonances 2 and 1 at 1.46
(broad s) and 1.15 ppm (broad s) are assigned to the CH2 and CH3 protons of the PIB chain, respectively. The 10, 9 CH2 and the 3, 4 CH3 resonances are assigned to the first and second terminal units of tert-Cl terminated PIB. Singlets (a), (b1), (b2), and (c) found at 1.99, 4.89, 4.68, and 1.82 ppm respectively, demonstrate the presence of olefinic groups; loss of HCl from PIB-Cl is well-documented.191 Integration of the olefinic protons indicated that about 30% of the PIB-OH had the olefinic end group shown in
Figure 4.26.
The 13C NMR spectrum is shown in Figure 4.27.
82 093006_PIBOH#15_CDCl3_13C.esp 9, 12, 10, 13, 23 6 10 13 16 19 22 25 15, 18 16, 19 CH3 CH3 CH3 CH3 CH3 CH3 CH3 458 9 11 12 14 15 17 18 20 21 23 24 1 C CH2 C CH2 C CH2 C CH2 C CH2 C CH2 C Cl
7CH2 CH3 CH3 CH3 CH3 CH3 CH3 2 3 10 13 16 19 m 22 25 OH 2 22 ’ 25’’ 11, 14, CH3 1 CH2 17 20 ’ 21 ’ 23’ PIB CH2 C CH2 C 24’ 25
CH3 21 CH3 22 ’ 25’ Impurity 23 22’ 3 CHLOROFORM-d 5 25’ 22 7 6 4 20’ 8 23’ 25’’ 24 21’ 24’ 20
145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 Chemical Shift (ppm)
Figure 4.27. 13C NMR spectrum of PIB-OH.
Assignments were based on previously published data.178,190 The aromatic protons of the initiating moiety are found at 145.75 (4), 128.54 (2), 127.26 (1) and 126.25 ppm
(3), respectively. The CH2 carbon 7 adjacent to the hydroxyl group shows up at 74.74 ppm. The other carbons neighboring the OH and Cl end groups are also found as single resonances: 59.26 ppm (23) and 53.11 ppm (8) for the methylene carbons, 71.67 ppm
(24) and 44.47 ppm (5) for the quaternary carbons, 35.60 ppm (25) and 14.53 ppm (6) for the methyl carbons. The IB repeat units carbons show up as three major resonances:
59.81 ppm for the methylene carbons, 38.43 ppm for the quaternary carbons, and 31.59 ppm for the methyl. The rest of the resonances demonstrate the presence of olefinic end groups in the PIB-OH chains.
83 The SEC chromatograms are shown in Figure 4.28. The light scattering signal shows the presence of some larger size matter, not apparent in the RI signal. This scatter is most probably due to some particulate contamination, such as TiO2 residues from the hydrolysis of the TiCl4 coinitiator. Mn = 5000 g/mol and Mw/Mn = 1.79 were obtained with using dn/dc = 0.108.
Figure 4.28. SEC traces (light scattering and differential refractometer detectors) of PIB-OH.
MALDI-TOF Mass Spectrometry (MS) is a particularly useful technique for determining chain end functionality of the polyisobutylene samples involved in this work.
In 2002, Ji et al.192 proposed a method to obtain MALDI-TOF mass spectra of isobutylene homopolymers that involved the conversion of the terminal double bond and/or tert-chlorine function into sulfonate groups in quantitative yield. They tested the 84 method on both monofunctional PIBs produced by living cationic polymerisation using
TMPCl (2-chloro-2,4,4-trimethylpentane) as initiator and commercial PIB samples. PIBs having sulfonate groups gave good MALDI-TOF mass spectra without the use of any additional ionization reagents; the spectra were generated using an all-trans-retinoic acid matrix.
In 2000, Kéki193 and co-workers reported MALDI-TOF of dihydroxyl telechelic polyisobutylene (HO-PIB-OH) synthesized from dichloro telechelic polyisobutylene made by living cationic polymerization that was then dehydrochlorinated followed by hydroboration/oxidation. 1,8-Dihydroxy-9(10H)-anthracenone (dithranol) was used as the matrix and silver trifluoroacetate (AgTFA, CF3COOAg) as the cationation agent. They obtained a spectrum containing two distributions of peaks. The main peaks were identified to be dihydroxyl-terminated PIB and the peaks at lower mass were attributed to monohydroxyl-terminated PIB. In 2002 the same authors194 offered a new synthetic method for the quantitative preparation of HO-PIB-OH starting from diolefinic PIB via epoxidation with dimethyldioxirane followed by conversion into aldehyde functional groups with zinc bromide and reduction of the aldehyde termini with LiAlH4 into primary hydroxyl groups. Based on the 1H NMR spectrum of the product obtained after the reduction, they concluded that the conversion was quantitative. The MALDI-TOF MS spectrum obtained using a dithranol/AgTFA matrix confirmed the presence of the dihydroxy end group. The authors claimed that the appearance of another series of peaks at the lower mass region of the spectrum was due to the in-source fragmentation of dihydroxy PIB under MALDI conditions. In 2004, they used the same type of matrix to
85 obtain the MALDI-TOF MS spectrum of bis(glucopyranosyl) polyisobutylene.195 They claimed the exclusive presence of this bifunctionalized PIB based on 1H NMR and
MALDI-TOF MS spectra.
The MALDI-TOF MS spectrum of our PIB-OH is shown in Figure 4.29. The analysis was carried out in the Department of Chemistry by David E. Dabney using dithranol as the matrix and silver trifluoroacetate as the cationation agent.
3.00 *
0 1500 2000 3000 4000 5000 m/z
Figure 4.29. MALDI-TOF MS spectrum of PIB-OH cationized with silver ions.
The mass difference between the neighboring molecular peaks is nominally 56 g/mol, which corresponds to the mass of the isobutylene repeat unit. Figure 4.30 shows the isotopic pattern of the oligomer marked with a star in Figure 4.29.
86 2.50
1530.8010
0 1520 1530 1540 1550 m/z Figure 4.30. An isotope cluster in the MALDI-TOF MS spectrum of PIB-OH.
The peaks ensemble in Figure 4.30 is a representation of a single isotope cluster of the respective n-mer. The m/z ([m/z]n) value of the monoisotopic peak is related to the mass of each fragment of the PIB molecule according to Equation (1).
[m / z]n = M I + nM IB + M end −group + M Ag (1) where MI is the mass of the initiator moiety, n is the number of isobutylene (IB), MIB is the mass of the IB unit (56.0626 g/mol), Mend-group is the mass of the end group, and MAg is the mass of the silver cation attached to the polymer chain (106.9051 g/mol). In the case of the n-mer in Figure 4.30, if we subtract the mass of the Ag cation from the observed m/z value of the monoisotopic peak (1530.8010 g/mol) and divide the result by the mass of the IB unit (56.0626 g/mol), we obtain the hypothetical value of n for this mer, i.e. 25.3983. If we multiply the digits after the decimal point (0.3983) by 56.0626, we obtain the mass value of the smallest end group possible for the PIB-OH molecule that is 22 g/mol. As this mass does not represent any probable structure for the function of the PIB sample characterized, we add 56 g/mol to the 22 g/mol mass previously 87 calculated as many times as needed to fit with a possible end group mass. In the case of this n-mer, adding three times 56 g/mol led to a 190 g/mol end group mass that corresponds to the PIB-OH structure shown in Figure 4.31.
Ag+
CH3 CH3 CH2
C CH2 C CH2 C
CH2 CH3 CH3 m OH
Figure 4.31. Structure of the PIB-OH molecule identified using MALDI-TOF MS.
Thus we can identify this n-mer as the 22-mer (the n = 25 previously calculated minus 3
IB units). No tertiary chloride was observed, which is consistent with the high probability of dehydrohalogenation under MALDI-TOF conditions. Figure 4.29 shows that the 22- mer is the first identifiable mer above the 1,000 g/mol cut-off mass although interference of the matrix and the silver are still observed. The cut-off mass is the value below which all ions are deflected in order to amplify higher mass ions in the spectrum. The purpose in this experiment is to minimize the matrix interference, which is generally between 700 and 1000 g/mol for dithranol. The other n-mers of the spectrum were identified in the way discussed above. The calculation gave the same end groups mass for each monoisotopic peak.
88 The procedure used for the synthesis of PIB-O-M is detailed in CHAPTER III,
3.3.9. Figures 4.32 and 4.33 show the 1H and 13C NMR spectra of the product obtained in
the reaction of PIB-OH with M-OTs.
110406_PIBether3_CDCl3_1H.esp 16 15 87 651 3 4 HC CH CH3 CH3 CH3 CH3 CH3 CH3 CH3 13 12 11 2 10 9 15 17 HC C C CH2 C CH2 C CH2 C CH2 C CH2 C CH2 C Cl
HC CH 14 CH2 CH3 CH3 CH3 CH3 CH3 CH3 16 m 16 15 765 1 3418 O
(b1)(b2) 18 CH2 CH2 19 (a) (b1) C PIB CH2 C 19 20 14 H2C CH3 CH3 17 (c) (e) CH3 (d) (b2) PIB CH C CH3 (e) (d)
7.14 2.70 0.52 2.00 1.76
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 Chemical Shift (ppm)
110406_PIBether3_CDCl3_1H.esp 2 1
3 5 (b1)(b2) CH2 (a)
PIB CH2 C
CH3 (c) (e) CH3 (d) PIB CH C 8
CH3 (e)
4 6 10 12 13 11 7 9 (c) (a) (e)
2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 Chemical Shift (ppm) Figure 4.32. 1H NMR spectrum of PIB-O-M.
89 The 1H NMR spectrum of the precipitated polymer did not show the presence of residual PIB-OH. The resonances of the CH2 protons adjacent to the hydroxyl group in
PIB-OH disappeared and a new signal assigned to the methylene protons adjacent to the novel end function was found at 3.33 ppm. New resonances appeared: the resonance 19
(s, 2H) assigned to the vinyl protons of the novel functional group shows up at 4.88 ppm and the CH2 protons 18 adjacent to the oxygen atom previously belonging to the tosylate model compound was also identified at 3.80 ppm (s, 2H). The resonance 20 of the methyl protons of the linked moiety can not be seen since it appears underneath the methyl protons signal of the PIB main chain.
110406_PIBether3_CDCl3_13C.esp 11, 14, 9, 12, 10, 13, 23 6 10 13 16 19 22 25 CH3 CH3 CH3 CH3 CH3 CH3 CH3 17 15, 18 16, 19 458 9 11 12 14 15 17 18 20 21 23 24 1 C CH2 C CH2 C CH2 C CH2 C CH2 C CH2 C Cl
7CH2 CH3 CH3 CH3 CH3 CH3 CH3 2 3 10 13 16 19 m 22 25 O
26CH2 22 ’ 25’’ CH3 CH2 C 27 20 ’ 21 ’ 23’ PIB CH C CH C 24’ H2C CH3 2 2 28 29 CH3 21 CH3 2 25’ 1 22 ’ 25 22’ 23 7 22 3 28 CHLOROFORM-d 5 29 6 26 20’ 8 20 27 24’ 25’’ 25’ 23’ 21’ 4 24
145 140 135 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 Chemical Shift (ppm)
Figure 4.33. 13C NMR spectrum of PIB-O-M.
90 Figure 4.33 shows the appearance of four resonance peaks (27, 28, 26 and 29 at
147.14, 112.00, 81.27 and 24.37 ppm, respectively) on the spectrum and the presence of a single resonance for the CH2 7. This confirms the quantitative conversion of PIB-OH into
PIB-O-M.
The SEC traces obtained (Figure 4.34) were similar to the ones obtained for the
PIB-OH sample. The same contamination as for PIB-OH was observed.
Figure 4.34. SEC traces (light scattering and differential refractometer detectors) of PIB-O-M.
The number average and weight average molecular weights and the molecular weight distribution of the PIB-O-M sample were respectively 4600 g/mol, 8100 g/mol and 1.77.
These data were calculated taking only the refractometer trace into account.
91 The MALDI-TOF MS spectrum of the PIB-O-M sample is shown in Figure 4.35.
It was obtained by using dithranol as the matrix and silver trifluoroacetate as the cationation agent.
2.00
0 3000 5000 1000 m/z Figure 4.35. MALDI-TOF MS spectrum of PIB-O-M cationized with silver ions.
The mass difference between the neighboring molecular peaks is nominally 56 g/mol, as expected. Figure 4.36 shows the observed cluster for the PIB-O-M 20-mer.
92
2
7
0
1.00 2 .
6
1
4 1
0 1412 1416 1420 1424 m/z Figure 4.36. Representation of the PIB-O-M 20-mer.
In addition, the calculations discussed in the case of the PIB-OH spectrum were made for each n-mer and the results agreed well with the structure expected for the PIB-
O-M (Figure 4.37).
Ag+
CH3 CH3 CH2
C CH2 C CH2 C
CH2 CH3 CH3 m
O
CH2
C
H2C CH3
Figure 4.37. Structure of the PIB-O-M molecule identified using MALDI-TOF MS.
The MALDI-TOF MS spectrum provides additional evidence of the quantitative preparation of the desired PIB-O-M model molecule.
93 4.2.4. Preliminary Model Experiments for the Synthesis of PIB-NPP from PIB-N
We found two pathways in the literature that could be used to synthesize the
pyrophosphate end of PIB-NPP starting from PIB-N. One was described by Chen et al. in
2002.181 They transformed 7-(2,6-dimethyl-8-hydroxy-2,6-octadienyloxy)-8-methyl-4-
trifluoromethyl-chromen-2-one (primary alcohol R-OH) into the corresponding
pyrophosphate using a brominated intermediate. The experimental conditions of the two
steps involved in this pathway are described in Table 4.1.
Chen’s procedure Step A Step B -OH -Br -Br -OPP - R-OH (0.17 g, 0.50 mmol) - R-Br (85 mg, 0.21 mmol)
- tetrabromomethane (0.20 g, 0.6 mmol) - (Bu4N)3.HP2O7.(0.55 g, 0.6 mmol)
- triphenylphosphine (0.16 g, 0.6 mmol) - solvent: dry acetonitrile (CH3CN)
- solvent: dichloromethane (CH2Cl2) - temperature: 27°C - temperature: 27°C - time: 6 h - time: 4 h - ion exchange column + - purification: flash chromatography (Dowex AG50X8, NH4 form) - yield: 80% - purification: preparative HPLC (eluant: acetonitrile and NH4HCO3 solution) - yield: 46%
Table 4.1. Chen experimental conditions for the synthesis of a pyrophosphate molecule.
In 2000, Coates et al.196 used a different procedure to synthesize geranyl
pyrophosphate. This two-step pathway includes a diethyl phosphate intermediate. The
conditions of the experiment are summarized in the following table (Table 4.2).
94 Preliminary experiments were carried out using model compounds. We tried to reproduce
the first step of the procedure described by Chen.
Coates procedure Step A Step B
-OH -OP(O)(OEt)2 -OP(O)(OEt)2 -OPP - geranol (200 mg, 1.30 mmol) - geranyl diethyl phosphate (180 mg, 0.62 mmol)
- pyridine (123 mg, 1.56 mmol) - (Bu4N)3.HP2O7.(H2O)3 (solid, 1.00 g, 1.05 mmol)
- diethyl chlorophosphate (236 mg, 1.37 mmol) - solvent: dry acetonitrile (CH3CN)
- solvent: dichloromethane (CH2Cl2) - temperature: RT - temperature: 0°C - time: 4 days - time: 30 min - ion exchange column + - purification: flash chromatography (Dowex 50 W-X8, NH4 form, 1.4 × 30 cm) - yield: ~90% - cellulose chromatography - storage at –20°C (2.4 × 15 cm, Whatman CF-11) - purification: preparative HPLC
(eluant: acetonitrile and NH4HCO3 solution) - yield: 78%
Table 4.2. Coates experimental conditions for the synthesis of geranyl pyrophosphate.
4.2.4.1. Bromination of the Hydroxyl Group of a Model Compound Standing for PIB-N
The model compound used was 3-methyl-2-buten-1-ol (Figure 4.38).
OH
Figure 4.38. Structure of 3-methyl-2-buten-1-ol.
95 Chen’s procedure181 was followed as detailed in CHAPTER III, 3.3.10. The spectra obtained for the crude product are shown in Figures 4.39 and 4.40. Only the 1H
NMR spectrum shows the presence of the desired product in the crude mixture.
1H eg.110 - CDCl3 - 25/07/05crude brominated model compound CBr4 PPh3 b CH 3 c H2 a C H C C Br 3 H d
b a
c
d
Integral 0.9439 2.0000 6.3072 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 (ppm)
Figure 4.39. 1H NMR spectrum of the brominated model intermediate obtained using Chen’s procedure.
96
eg.110 - CDCl3 - 25/07/05crude brominated model compound CBr4 PPh3
134.3311 132.7599 132.7308 132.3380 132.2362 130.7960 130.5705 130.4396 129.7486 128.9339 128.8102 120.7945 29.8939 25.8570 17.6158 9.9928
140 130 120 110 100 90 80 70 60 50 40 30 20 10 (ppm)
Figure 4.40. 13C NMR spectrum of the brominated model intermediate obtained using Chen’s procedure.
97 To purify this crude mixture, the flash chromatography technique on silica gel was used with 100% acetonitrile eluent but was not successful. The product had not been identified in terms of chromatographic fraction; i.e., no pure product could be isolated.
Another trial should be made. The crude mixture was not soluble in most of the common solvents (dichloromethane, toluene, ethyl acetate…). Only a 100% acetonitrile eluent did both solubilise the mixture and separated the two darkest marks visible on the TLC plates.
Thibaut Forestier, a French Master Student working under the supervision of Pr.
Alain Deffieux in LCPO in 2005, carried out the same experiment. He wrote in his lab book that he used another method to purify the crude mixture. He first filtered it to remove the excess PPh3 and CBr4, and then concentrated it. Finally, he “cryodistilled” it to obtain the pure product in 95% yield. Cryodistillation consists of the usual vacuum distillation setup that cooling system is undergone using liquid nitrogen clamps on the collecting glassware. We suggest that this method of purification of the brominated intermediate should be tried in a future trial.
4.2.4.2. Synthesis of Tris(tetra-n-butylammonium) Hydrogen Pyrophosphate
In preparation for the synthesis of the pyrophosphate end, we prepared tris(tetra-
182 n-butylammonium) hydrogen pyrophosphate [(NBu4)3HP2O7] using the procedure described in CHAPTER III, 3.3.11. The 1H, 13C and 31P NMR spectra are shown in
APPENDIX D. All peaks were identified in the 1H and 13C NMR spectra and integration
98 of these peaks corresponds exactly to the ratios expected from the chemical formula of
31 (NBu4)3HP2O7, demonstrating that pure product was obtained. The P NMR spectrum was a little less convincing and a doubt remains. Indeed the literature claims a singlet at -
1.60 ppm and we found two, a small one at 1.09 ppm and a tall one at -8.39 ppm. The exact same spectra were obtained by Forestier when he carried out the experiment following the exact same procedure.
This solid compound is extremely hygroscopic. Thus it is important to store this salt in a desiccator over calcium sulfate at -20°C in order to preserve it for several months. It can also be stored in a dry box for several weeks. The water content of the
1 hygroscopic solid can be determined by H NMR in benzene-d6. The hydrate can be partially dehydrated immediately before use by repeatedly dissolving the material in anhydrous acetonitrile and removing the solvent under vacuum at room temperature.
The last step involving this salt and the brominated PIB-N will be the synthesis of the pyrophosphate end. It should be tried in the future. The solvent used in the literature was acetonitrile. It might be a concern since PIB is not soluble in that solvent, but a two- phase reaction could be successful. Otherwise, the solvent should be changed. It is known that tris(tetra-n-butylammonium) hydrogen pyrophosphate is highly soluble in chloroform, dichloromethane, tetrahydrofuran, benzene,182 which are also known as PIB solvents.
99 CHAPTER V
CONCLUSION
One of the objectives of this thesis was to critically review research aimed at the synthesis of natural rubber (NR). We analyzed information pertaining to the biosynthesis of NR in the biochemical literature, and translated it into polymer chemical formalism, i.e., initiation, propagation and termination. We found that this biosynthesis proceeds by a chain growth (addition) mechanism that fits the definition of living polymerization set forth by the IUPAC, and that all the species identified by earlier authors that are known to arise during biosynthesis can be described in terms of carbocationic intermediates. A combination of these two critical parameters, livingness and carbocationic intermediates, led us to propose that the biosynthesis of NR proceeds by a natural living carbocationic polymerization mechanism (NLCP), in which the dormant pyrophosphate chain ends are reversibly activated by the rubber transferase enzyme and divalent cation cofactors.
Another goal of this research was to design a feasible synthetic pathway to yield a macroinitiator for the future synthesis in vitro of a novel polyisobutylene-block-cis-1,4- polyisoprene (PIB-b-NR) diblock copolymer. PIB-OH samples made by “traditional” living carbocationic polymerization using a α-methylstyrene epoxide (MSE)/TiCl4 system to initiate isobutylene polymerization, were provided to us. The strategy developed for the synthesis of the target PIB-NPP macroinitiator consisted of six main 100 steps. Three of those were successfully completed. The others were studied by carrying out experiments using model compounds, and the results obtained were extremely promising. Additional experiments are still needed to synthesize the ultimate macroinitiator, and in vitro reactions could then be carried out to make the diblock copolymer targeted.
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APPENDICES
119 APPENDIX A
STRUCTURE OF THE ADENOSINE TRIPHOSPHATE MOLECULE
Structure of the Adenosine TriPhosphate molecule.
ATP consists of a base (adenine), a sugar molecule (ribose) and a phosphate chain.
Remarks
ATP works by losing the endmost phosphate group when instructed to do so by an enzyme.
The ATP molecule acts as a chemical “battery”, storing energy when it is not needed, but able to release it instantly when the organism requires it.
Source: http://www.bris.ac.uk/Depts/Chemistry/MOTM/atp/atp1.htm; visited on 6/26/2006 120 APPENDIX B
STRUCTURE OF THE NADP+ MOLECULE
NADP+ molecule
121 APPENDIX C
NMR SPECTRA OF PIB-OH ETHERIFIED
WITH A METHYLPROPENE MODEL COMPOUND
eg.104 - CD2Cl2 - 21/06/2005PIB-O-(model compound)
2.10 2.00 1.90 1.80 1.70 1.60 1.50 1.40 1.30 1.20 1.10 1.00 0.90 0.80 0.70 (ppm) 1H NMR spectrum of PIB-OH etherified with a methylpropene model compound.
eg.104 - CD2Cl2 - 21/06/2005PIB-O-(model compound)
Integral 2.0000 1.9180 10.337 5.5586 2.3672 3.1512 7.6 7.4 7.2 7.0 6.8 6.6 6.4 6.2 6.0 5.8 5.6 5.4 5.2 5.0 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 (ppm) 13C NMR spectrum of PIB-OH etherified with a methylpropene model compound.
122 APPENDIX D
NMR SPECTRA OF TRIS(TETRA-N-BUTYLAMMONIUM)
HYDROGEN PYROPHOSPHATE
1H eg.068 - D2O - 22/07/04 d c b a O O CH CH CH CH H 2 2 2 3 CH CH CH CH O P P ON 2 2 2 3 CH CH CH CH O 2 2 2 3 O O CH2CH2CH2CH3 N N Bu Bu Bu Bu Bu Bu Bu Bu
a
b d c
Integral 23.567 24.003 24.196 36.000 4.8 4.6 4.4 4.2 4.0 3.8 3.6 3.4 3.2 3.0 2.8 2.6 2.4 2.2 2.0 1.8 1.6 1.4 1.2 1.0 0.8 (ppm)
1H NMR spectrum of tris(tetra-n-butylammonium) hydrogen pyrophosphate [(NBu4)3HP2O7].
123
13C eg.068 - D2O - 22/07/04
58.0328 58.0110 52.5702 25.1189 23.0531 19.0743 12.7534 a d c b a O O CH CH CH CH H 2 2 2 3 CH CH CH CH O P P ON 2 2 2 3 CH CH CH CH O 2 2 2 3 O O CH2CH2CH2CH3 c N N Bu Bu Bu Bu Bu Bu Bu Bu b
d
60 58 56 54 52 50 48 46 44 42 40 38 36 34 32 30 28 26 24 22 20 18 16 14 12 (ppm)
13C NMR spectrum of tris(tetra-n-butylammonium) hydrogen pyrophosphate [(NBu4)3HP2O7].
eg.068 - D2O - 22/07/04
1.0902 -8.3914
2.0 1.0 0.0 -1.0 -2.0 -3.0 -4.0 -5.0 -6.0 -7.0 -8.0 -9.0 -10.0 -11.0 -12.0 (ppm)
31P NMR spectrum of tris(tetra-n-butylammonium) hydrogen pyrophosphate [(NBu4)3HP2O7].
124