Chemical Reviews - March 2010

Chem. Rev. 2010, 110, 12131232

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Spore Photoproduct: A Key to Bacterial Eternal LifeCeline Desnous, Dominique Guillaume, and Pascale Clivio*, ICSN, UPR CNRS 2301, 1 Avenue de la Terrasse, 91198 Gif sur Yvette Cedex, France and UMR CNRS 6229, 51 Rue Cognacq Jay, 51096 Reims Cedex, France Received June 1, 2007

Contents1. Introduction 2. Sporulation and Its Implications 3. Major UV-Induced DNA Photoproducts 3.1. Direct UV Radiation Absorption by DNA Nucleobases 3.1.1. Cycloaddition Reactions 3.1.2. Photohydration Reactions 3.2. UV-A-Induced Photosensitization 3.2.1. Energy Transfer from a Photosensitizer 3.2.2. Type I Photosensitization 3.2.3. Type II Photosensitization 3.3. DNA Damage Caused by As Yet Unidentied Mechanisms 3.3.1. Guanine Oxidation 3.3.2. Strand Break Formation 4. UV-C-Induced DNA Photoproduct Formation in Bacteria 4.1. In Bacterial Vegetative Cells 4.2. In Bacterial Spores 4.3. Stereochemical Consideration of SPDNA Formation 4.4. Formation Mechanism of SPDNA 5. Factors Inuencing SPDNA Formation in Spores 5.1. Small Acid-Soluble Spore Proteins 5.2. Dipicolinic Acid 5.3. Pressure/Hydration Level 5.4. Temperature 5.5. Conclusions 6. Articial Production of SPDNA, SPTIDE, and SPSIDE 6.1. UV Irradiation of Bacterial Vegetative Cells 6.2. UV Irradiation of Isolated DNA 6.2.1. Isolated DNA 6.2.2. Isolated DNA Complexed with R/-Type SASP 6.2.3. Isolated DNA in the Presence of Ca-DPA 6.2.4. Isolated DNA in the Presence of R/-Type SASP and Ca-DPA 6.3. UV-C Irradiation of Oligodeoxynucleotides 6.4. Irradiation of Thymidine 6.4.1. By Direct 254 nm Irradiation 6.4.2. By Photosensitization 6.4.3. By -Irradiation, Heavy Ion, or Electron Bombardment 1213 1215 1215 1215 1215 1216 1217 1217 1217 1217 1218 1218 1218 1218 1218 1218 1219 1219 1220 1221 1222 1222 1222 1222 1223 1223 1223 1223 1224 1224 1225 1225 1225 1225 1225 1225

7. Chemical Synthesis of SP and Derivatives 7.1. Synthesis of SP 7.2. Synthesis of SPSIDE and SPTIDE 8. DNA Photoproduct Repair 8.1. Conventional DNA Photoproduct Repair 8.1.1. Nucleotide Excision Repair (NER) 8.1.2. Base Excision Repair (BER) 8.1.3. Photolyase-Induced Repair 8.1.4. Repair of DNA Strand Breaks 8.1.5. Others 8.2. SPDNA Repair 9. Conclusions 10. Abbreviations 11. Acknowledgments 12. References

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1. IntroductionThe cells of all living organisms are continuously exposed to a large variety of harmful agents that can induce either temporary or permanent deleterious effects. In response to these genotoxic stresses, only those cells endowed with efcient defense mechanisms have been able to overcome lifes challenges and evolutionary demands and survive. By directly targeting DNA nucleobases, solar UV radiation is a ubiquitous and particularly potent physical agent capable of altering the genome integrity. The most deleterious UV wavelengths are those in the 200-280 nm range (UV-C). Fortunately for humanity, although UV-C permeates space, it does not in fact reach the surface of the earth thanks to the presence of ozone and other protective layers high in the atmosphere. Nevertheless, there are important industrial uses for articially produced UV-C, mostly exploiting its germicidal properties. Though UV radiation-induced DNA damage (UV damage or photodamage) has had an indisputable evolutionary function, from a conservative standpoint the only cells able to transmit their genetic material unaltered are those capable of minimizing the frequency of the photodamage and/or those possessing accurate DNA repair pathways. The frequency of photodamage occurrence can be attenuated by extranuclear constituents. In addition, inaccurate damage repair can result from two distinct phenomena: either overwhelming photodamage or a decient DNA damage repair system. Both these phenomena can be induced by the long-term UV exposure of DNA. Once the cell chromosomal material has been UV damaged, the cell cycle often undergoes dramatic modication. In humans, unrepaired photodamage can lead to mutation and

* To whom correspondence should be addressed. Phone: (33) 326 918 027. Fax: (33) 326 918 029. E-mail: [email protected]. ICSN, UPR CNRS 2301. UMR CNRS 6229.

10.1021/cr0781972 2010 American Chemical Society Published on Web 11/05/2009

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Celine Desnous, born in Toulouse, France (1979), graduated in 2002 from the Ecole Nationale Superieure de Chimie de Paris. In 2005, she received her Ph.D. degree in Organic Chemistry from the University of Orsay (Paris XI), working under the supervision of Dr. P. Clivio on spore photoproduct chemistry. She is now working as a research engineer for the worlds leading manufacturer of technical and creation papers ArjoWiggins, in one of its research centers based in Apprieu, France.

Pascale Clivio studied Pharmaceutical Sciences at the University of Bourgogne in Dijon between 1980 and 1985. In 1989, she obtained her Ph.D. degree in Natural Product Chemistry at the University of Reims Champagne Ardenne (URCA) under the supervision of Professor Monique Zeches. In 1989, she got a permanent research position at CNRS (ICSN, Gif sur Yvette) to work with Dr. Jean-Louis Fourrey in the eld of nucleic acids chemistry and photochemistry. In 1997, she received a one-year fellowship from the International Agency for Research on Cancer to work with Professor John-Stephen Taylor at the Washington University in Saint Louis, Missouri. She recently moved to the FRE CNRS 2715, URCA. Her current research interests include the study of conformational impact on DNA photochemistry, the chemistry of DNA repair, and the synthesis of DNA photoproducts for biological studies. Another area of research interest is the synthesis of fat nucleosides.

Dominique Guillaume was born in France in 1959. He obtained his PharmD degree in 1983 and Ph.D. degree in 1986 from Universite de Reims Champagne-Ardenne (URCA). He spent 1986-1988 as a postdoctoral fellow in the laboratory of Daniel H. Rich at UW-Madison and became an assistant professor in 1988 at the school of pharmacy, Universite Paris V, France. He was a visiting faculty member in 1997 at Washington University in Saint Louis. In 1999, he became Full Professor of Medicinal Chemistry and moved to URCA in 2005, where his research eld includes peptide and nucleic acid chemistry. He has authored, or coauthored, more than 80 publications.

cancer; premature cell apoptosis is another frequent outcome. In the vegetal kingdom, another frequently observed consequence is growth reduction. The equilibrium of fragile terrestrial ecosystems can be strongly affected in the long term. Similarly, UV damage affecting fungi, bacteria, or phytoplankton can lead to changes in soil microbial communities, the biomass, and in aquatic ecosystems. Bacterial spores represent a curious and fascinating lifeform. Their resistance to UV-C radiation is much higher than that of their corresponding vegetative forms.1 In addition, bacterial spores conserve their capacity for germination for an extremely long time.2,3 Hence, concerns regarding the efciency of UV-C-assisted sterilization4,5 and, consequently, the actual usefulness of UV-C in controlling the biological safety6 of food and the subsequent minimization of the bioterrorism threat7 have recently been addressed.

The potentially elevated risk of bacterial planetary contamination through space exploration is also currently of great interest.8,9 Spores also have to face UV-A and -B radiation in order to survive. The deleterious effect of UV on spores is a consequence of either direct effects on their DNA components or the results of the formation of reactive oxygen species (ROS). Bacterial spore resistance to UV-A and -B is attributed mainly to the presence of melaninlike absorbing pigments in the spore outer layers. Bacterial spore resistance to UV-C radiation has been associated with the unique photochemical behavior of its DNA. In vegetative bacterial cells, UV-C radiation induces DNA damage principally through pyrimidine dimerization, which produces cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts ((6-4) PPs). In contrast, in bacterial spores a single and totally different photoproduct, 5-(R-thyminyl)-5,6-dihydrothymine, is produced by UV-C irradiation. During the 5 years between the initial isolation of this photoproduct and its structural elucidation, it was simply called the spore photoproduct or SP, and this name is still commonly used. Damage can be repaired during germination through the action of a specic enzyme named SP lyase. Formation of the spore photoproduct and its subsequent ability to undergo such efcient repair explains the low vulnerability of bacterial spores to UV-C exposure. Particularly during the last 15 years,10-17 numerous reviews have covered various aspects of the UV resistance of spores. Only the spore photoproduct itself has not been the subject of its own review. This is particularly regrettable in light of the pivotal involvement of this photoproduct in the strong UV-C resistance of bacterial spores and the importance of its chemistry in helping to decipher DNA-specic behaviors. Hence, this review will focus on the chemistry of the spore photoproduct from its formation to its repair and including its chemical synthesis.

Spore Photoproduct

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2. Sporulation and Its ImplicationsIn response to environmental conditions such as nutrient depletion, some microorganisms initiate a unique survival process called sporulation.18-20 This process begins with the formation of a predivisional cell whose gene expression is governed by the Spo0A protein. This is followed by an asymmetric division. As a result, a large mother cell and smaller cell called a forespore are formed. Each has its own individual but interconnected gene expression program. The forespore, which will become the core of the mature spore, acquires a second membrane through engulfment by the mother cell. The space between the two membranes is later lled in by cell wall material to give the spore cortex. Finally, an outer shell of protein (the coat) is laid on the external surface of the developing spore to provide an additional protective layer. Meanwhile, during its maturation, the forespore undergoes two additional major transformations. Both of these are of the utmost importance for the protection of its DNA: (1) dehydration, due in part to the uptake of pyridine-2,6-dicarboxylic acid (dipicolinic acid, DPA) from the mother cell, and (2) compacting of its chromosomal material by a family of small, acid-soluble spore proteins (SASP). In the nal step, the mature spore is released into the environment through lysis of the mother cell. Spores represent a unique form of life since most of them possess only a single chromosome and display no apparent metabolism, a state dened as cryptobiotic. Spore dormancies of longer than 25 million years3 and possibly even 250 million years21,22 have been reported. Despite their apparent lack of life some spores play an essential ecological and environmental role.23 Not all bacteria genera possess the ability to sporulate. The spore-forming Bacillus (and to a lesser extent Clostridium) species are the most studied. Observations of this species have often been generalized and extended to other spore-forming genera. However, species with extreme UV resistance have been recently isolated.24 This clearly supports the existence in the spores of some bacterial species with specic biochemical processes much more complex than previously anticipated. Under appropriate circumstances, spore dormancy ends and the spore germinates25 into a vegetative cell whose genome, despite the possible accumulation of DNA UV damage during dormancy, is preserved to an amazing extent relative to the genome of the mother cell.

3.1. Direct UV Radiation Absorption by DNA NucleobasesBoth UV-C and -B photon absorption give rise essentially to cycloaddition and photohydration reactions. Pyrimidine nucleobases (thymine and cytosine) are the most sensitive to UV-B and -C radiation.

3.1.1. Cycloaddition ReactionsAt the dipyrimidine sites (1-4) in DNA, cyclobutane pyrimidine dimers (CPDs) and (6-4) pyrimidine pyrimidone photoproducts ((6-4) PPs) are principally formed by direct photon absorption (Figure 1). CPDs are the most abundant dipyrimidine photoproducts. They result from a [2 + 2] cycloaddition reaction between the C5-C6 double bonds of two adjacent pyrimidines (Figure 1). Depending on the nature of the C4 (O or NH) and C5 (CH3 or H) pyrimidine substituent, four different PPs (5-8) are formed. These PPs consequently can have a cissyn stereochemistry since pyrimidines in DNA adopt an anti glycosidic bond conformation. Cytosine-derived CPDs (6-8) are unstable and spontaneously deaminate at their C4 position, thus leading to secondary photoproducts belonging to the uracil-derived CPD family. Under UV-C radiation, CPDs can revert to their parent nucleobases.

Figure 1. UV-induced c,s CPDs in DNA.

3. Major UV-Induced DNA PhotoproductsUV radiation is arbitrarily divided in three wavelength domains: UV-C encompasses the 220-280 nm, UV-B the 280-320 nm, and UV-A the 320-380 nm range. DNA absorbs mainly in the UV-C and to a smaller extent in the UV-B range. It barely absorbs in the UV-A domain. Consequently, only UV-B and -C have any direct effect on DNA. However, UV-A radiation is capable of indirectly damaging DNA through the participation of a photosensitizer. In this review, we will focus only on DNA photoproducts formed by low-intensity continuous UV irradiation that mostly involves the rst excited states of the nucleobases. This is in contrast to the high-intensity radiation delivered by lasers which produces nucleobase radical cations. Numerous reviews have already covered the photochemistry of DNA.26-34 In this section, we will only discuss the major classes of DNA photoproducts. Further references can be found in the reviews mentioned above.

(6-4) Pyrimidine pyrimidone photoproducts are the second most abundant PPs formed in DNA. They derive from a sequencespecic Paterno-Buchi reaction between the C5-C6 double bond of the pyrimidine on the 5 side and the exocyclic double bond at the C4 atom of the pyrimidine on the 3 side, i.e., the carbonyl of a thymine residue or the imine tautomeric form of a cytosine residue (Figure 2). The resulting four-membered ring intermediate (oxetane or azetidine) is unstable and undergoes a ring-opening reaction leading to (6-4) PPs (9-12) (Figure 2). Formation of the four-membered ring intermediate was ascertained using the photochemistry of thio-nucleobases that lead to a more stable thietane intermediate.35 (6-4) PPs 11 and 12 involving a cytosine at their 5 end can spontaneously deaminate, leading to secondary photoproducts. After UV-A or -B absorption, (6-4) PPs can be converted into their corresponding Dewar (Dwr) valence isomer (13-16) (Figure 3). This latter adduct can revert to its (6-4) isomer under UV-C radiation. Under prolonged UV-C exposure, the (6-4) pyrimidine pyrimidone motif can undergo a ring contraction reaction, leading to a 2-imidazolone (5-4) pyrimidone derivative.36


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Figure 2. UV-induced (6-4) PPs (9-12) in DNA.

Sequence-specic photoaddition of adenine residues with vicinyl thymine residues can also occur. Thus far only the ring-expanded derivative 23 produced from the TA site 21 has been isolated. Derivative 23 results from ring opening of the cyclobutane adduct 22 generated by UV-C-induced cycloaddition between the C5-C6 double bond of the 5end thymine residue and the C5-C6 double bond of the 3end adenine residue (Figure 5). The X-ray structure of the TA adduct at the dinucleotide level is now available.37

3.1.2. Photohydration ReactionsPhotohydration reactions usually involve pyrimidine bases. Hydration results in the saturation of the C5-C6 double bond and in the nonstereoselective substitution of the C6 atom by a hydroxyl group. Cytosine residues are the most prone to this kind of photoreaction. The resulting unstable cytosine hydrate derivative 24 can either revert through dehydration to the starting compound or deaminate to afford the uracil hydrate residue 25. The latter dehydrates much less readily than 24 (Figure 6).38,39 The UV-C-induced hydration reaction of thymine and purine residues in DNA leading, respectively, to 5-hydroxy-5,6dihydrothymine and 4,6-diamino-5-formamidopyrimidine or 2,6-diamino-4-hydroxy-5-formamidopyrimidine derivatives has been reported.40 However, these results have been contested.32

Figure 3. UV-induced Dewar PPs in DNA.

To a minor extent, purine nucleobases are also photoreactive. However, only adenine has been shown to give rise to cycloaddition reactions with adjacent nucleobases in DNA. Under UV-C irradiation the unstable azetidine 18 is generated at diadenine sites (17) by a cycloaddition reaction between the N7-C8 double bond of the 5-end residue and the C5-C6 double bond of the 3-end adenine residue (Figure 4). Azetidine 18 can then either spontaneously rearrange to afford 19 or undergo a hydration reaction, ultimately leading to 20 (Figure 4).

Figure 4. Formation of UV-induced AA PPs in DNA.

Spore Photoproduct


Figure 5. Formation of UV-induced TA photoproduct 23 in DNA.

3.2.2. Type I PhotosensitizationOne-electron transfer from DNA nucleobases to the excited photosensitizer (Type-I photosensitization) can lead to nucleobase radical cations that can subsequently undergo either hydration or deprotonation. In DNA, guanine is the nucleobase most prone to one-electron oxidation. This can be directly attributed to the fact that guanine has the lowest ionization potential or indirectly because transfer through the DNA of a positive charge originating from another nucleobase radical cation can occur. Consequently, adenine and pyrimidine nucleobases are less susceptible to this photochemical process. Nevertheless, adenine and pyrimidine nucleosides can be targeted by a one-electron oxidation process. 3.2.2.1. Oxidized Purines. Hydration of the guanine radical cation 26 leads to the formation of the 8-hydroxy7-yl-guanine oxidizing radical 27. Upon reduction this in turn yields 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua, 28) in DNA (Figure 7). Oxidation of 27 gives the 8-oxo7,8-dihydroguanine derivative (8-oxoGua, 29). Deprotonation of 26 leads to an oxazolone derivative 30 through a complex cascade of reactions. 3.2.2.2. Oxidized Pyrimidines. In DNA, the one-electron oxidation of pyrimidines is a minor pathway that ultimately leads to the formation of 5-(hydroxymethyl)uracil, 5-formyluracil, barbiturate, as well as hydantoin residues.

Figure 6. Hydrolytic deamination of cytosine hydrate derivative 24 in DNA.

In summary, pyrimidine as well as purine nucleobases are susceptible to UV radiation, and consequently, several types of photoproducts can be formed through radiation exposure of isolated DNA. However, in cellular DNA, CPD and (6-4) PPs are by far the most frequently produced adducts. Photohydrates, on the other hand, are generated in much smaller quantities. The formation of purine photoproducts in cellular DNA is yet to be conrmed.

3.2. UV-A-Induced PhotosensitizationUV-A damage of DNA is an indirect effect arising from the action of so-far unidentied photosensitizers in cells. Three general pathways can lead to the formation of DNA photoproducts.

3.2.1. Energy Transfer from a PhotosensitizerOnce excited by the UV-A radiation, the photosensitizer can directly transfer its energy to DNA, most likely via a triplet energy transfer mechanism. This type of reaction affects mainly dithymine sites producing as the major photoproduct c,s T[CPD]T (5). The cytosine-derived photoproducts c,s T[CPD]C (6) and c,s C[CPD]T (7) are generated in lower yields. It should be noted that this excitation process does not afford (6-4) PPs since they result from singlet-state excitation.

3.2.3. Type II PhotosensitizationFinally, the excited photosensitizer can also generate singlet oxygen (type II photosensitization) that further reacts with nucleobases in the DNA to give oxidized products. Only guanine residues have been shown to be susceptible to this mechanism, producing the 8-oxoGua derivative (29) (Figure 8).

Figure 7. UV-A-induced guanine-oxidized PPs in DNA by type I photosensitization.


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Figure 8. UV-A-induced 8-oxoGua formation in DNA by type II photosensitization.

3.3. DNA Damage Caused by As Yet Unidentied Mechanisms3.3.1. Guanine OxidationExposure of DNA to UV-B or -C radiation also results in the formation of some of the 8-oxoGua derivative 29. The mechanism of its formation could involve either the generation of ROS or a one-electron oxidation. This kind of photoinduced oxidation reaction is, however, a minor process compared to the formation of CPDs in these wavelength domains.

3.3.2. Strand Break FormationDNA strand breakage (single-strand breaks (SSBs) and double-strand breaks (DSBs)) is also observed after exposure to UV-A, -B, and -C radiation,28,41-43 although it is a minor process compared to the formation of CPDs. Possibly, UV-A and -B irradiation triggers a complex oxidative process involving the production of the highly reactive hydroxyl radical by the mitochondria and type I photosensitization which ultimately leads to DNA strand breakage.34,44 Strand breaks following UV-C irradiation would originate from a different mechanism involving the excited sugar phosphate backbone.45

4. UV-C-Induced DNA Photoproduct Formation in Bacteria 4.1. In Bacterial Vegetative CellsIn bacterial vegetative cell DNA, CPDs and (6-4) PPs are the major lesions formed as a result of UV-C irradiation (Figure 1).26,27,46 Not all of these PPs are generated with the same efciency: the major adducts formed are the c,s T[CPD]T 5, T[6-4]C 10, and c,s T[CPD]C 6 and/or the c,s C[CPD]T 7.27,46 In B. subtilis vegetative cells, for a UV dose of 0.1 J/cm2, c,s T[CPD]T 5 formation involves 5% of the total amount of thymine residues.11

residues from different DNA strands represents less than 1% of the intrastrand SP for a UV dose of 1 J/cm2.48 In the DNA of B. subtilis spores, for a UV-C dose of 0.1 J/cm2, spore photoproduct formation involves 7.5% of the total amount of thymine residues while c,s T[CPD]T (5) represents less than 0.2%.11 Interestingly, when spores of B. subtilis are exposed to both UV-B and -A radiation, spore photoproduct formation still remains the major event even though production is much lower (a 103- and 106-fold decrease, respectively) compared to 254 nm irradiation.49,51 Strictly speaking, the term SP refers exclusively to the dipyrimidine nucleobase structure 31. However, the term is frequently used rather loosely to refer to diastereomeric mixtures in which the N1 atom of each nucleobase is substituted. In this review, the acronym SP will be used exclusively for the nucleobase dimer structure 31. Acronyms SPSIDE and SPTIDE will be used for the dinucleoside analogue (32) and dinucleotide analogues (33, 34) of SP, respectively. The term ias-SPDNA will be used for damaged oligodeoxynucleotides (ODNs) or DNA 35 and 36 containing at least one intrastrand SP-type lesion, and the term irs-SPDNA will describe damaged ODNs or DNA 37 and 38 containing at least one interstrand SP-type lesion. If the intra- or internature of the spore photoproduct in DNA is not known, the acronym SPDNA will simply be used.

4.2. In Bacterial SporesAfter UV-C irradiation only very small amounts of CPDs, (6-4) PPs, and single- and double-strand breaks are produced in spore DNA. Some indirect evidence derived from the slight UV sensitivity of nonhomologous end joining repair mutants has recently conrmed the minor formation of strand breaks induced in spore DNA.47 For a UV dose of both 148 and 1.6 J/cm2,49 the cumulative effect of CPDs, (6-4) PPs, and single- and double-strand breaks represents less than 1% of all the DNA damage in the spores. Since this UV dose range far exceeds the minimum dose required to kill 90% of spores, the physiological effects of CPDs, (6-4) PPs, and single- and double-strand breaks are considered negligible.49 The PP formed in spore DNA is almost exclusively the 5-(Rthyminyl)-5,6-dihydrothymine (or spore photoproduct (SP)).50 It arises almost entirely from two adjacent thymidine residues on the same DNA strand. SP formation between thymidine

Spore Photoproduct


4.3. Stereochemical Consideration of SPDNA FormationThe chiral compound SP (31) was isolated in 1965 following the acid hydrolysis of SPDNA.50 Since the hydrolytic step resulted in the loss of the optically pure sugar moiety, no mention of the enantiomeric purity of the SP-5,6dihydrothymine moiety C5 position was given.52 Four isomers of ias-SPDNA (35 and 36, Figure 9) can be expected within the DNA strand depending on (1) the glycosidic bond conformation of the parent thymines at the time of the C5-CH2 bond formation and (2) the 5-end or 3-end location of the thymine residue to be saturated. Theoretically, each ias-SPDNA can result from two sets of adjacent thymidine conformers (Figure 9). The conguration of the 5,6-dihydrothymine residue C-5 atom is dictated by the syn/anti conformation of the glycosidic bond of its thymidine precursor, while the 5-R-thyminyl moiety can result from a syn or anti glycosidic bond conformation of the corresponding thymidine. The formation of ias-SPDNA within the double helix is likely to be regio- and stereospecic since SPTIDE is observed as a single peak in the HPLC-MS/MS chromatogram of enzymatically digested SPDNA.53 In the A- or B-DNA double helix the glycosidic bond conformation of the nucleobases is in the anti domain. In the dry state, DNA adopts an A conformation. Thus, since SPTIDE isolated from spore DNA and from UV-C-irradiated dry DNA display indistinguishable HPLC and mass fragmentation patterns,48 the nucleobases

in the DNA of spores are most likely in the anti glycosidic bond conformation domain. Therefore, only 35b and 36a can be formed. Recent experimental results using 32 as a model substrate for repair studies have identied the C5 S isomer of 32 as being diastereoselectively repaired by the SP lyase.54,55 This result has led to the suggestion of a C5 S conguration in ias-SPDNA, and consequently, the dihydrothymine moiety location should be at the 3 end of the dinucleotide motif as in 36a (Figure 9). However, extensive NMR studies carried out on SPTIDE have recently demonstrated that the dihydrothymine moiety is located at the 5 end of the dinucleotide motif and that its C5 conguration is R.56 Therefore, 35b is the correct structure for ias-SPDNA. Repair experiments consistent with this result have also been reported.57 Thus, ias-SPDNA results from bond formation between the methyl group of the 3-end thymine and the C5 atom of the 5-end thymine residues.

4.4. Formation Mechanism of SPDNATwo mechanisms are currently envisioned to explain SPDNA formation after UV-C irradiation. The methylene link formation could follow a reaction between two close in space but independently UV-generated thymine-derived radicals: the 5-R-thyminyl radical (37) and the 5,6-dihydrothymin-5-yl radical (38) (Scheme 1, path A).52 Indeed, evidence has been given for the possible formation by UV of these two different types of radicals from thymine.58-60 Nevertheless, the simultaneous formation of these two

Figure 9. Possible glycosidic bond conformers at dithymine sites in DNA and induced structures of ias-SPDNA isomers.

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different types of radicals at dithymine sites appears to be an event of low probability. In addition, neither the oxidative products derived from 37 and 38 or the dimerized adducts derived from two identical radicals have ever been isolated. The second hypothesis was proposed after analyzing photolysis experiments using methyl-deuterated thymidine. These experiments suggested a concerted mechanism involving the methyl group of one thymidine and the double bond of the second thymidine (Scheme 1, path B).26 However, neither of these two hypotheses explains the respective role or difference in photoreactivities of the thymine residues (at either 5 or 3) in ias-SPDNA formation. Nor is the inuence of the pyrimidine C4 substituent fully explained, since no cytosine-derived SP-like PPs have ever been isolated either in vivo or in vitro even though in the latter case a large range of conditions has been studied. Excited thymine residues can also be generated by photosensitization. Benzophenone and pyridopsoralens61,62 as well as the physiologically relevant calcium dipicolinate55,63,64 have all been identied as photosensitizers capable of performing this function.

of PPs per 104 bases as a function of the UV dose. It is important to note that the calculated values have no experimental signicance when the dose unit lies outside the range of the linear part of the curve. Clearly, a comparison between the two methods is only possible if the sequence of the irradiated DNA is known and if the quantitative results expressed as percent of thymine have been calculated using the linear part of the curve reporting the number of PPs per 104 bases as a function of the UV dose. There are several limitations that need to be considered when comparing quantitative data since they depend on both the DNA concentration and the specic experimental setup used for irradiation. In addition, some early studies reported the amount of PPs formed for a single UV dose. However, since damage formation is not necessarily a linear function of the UV dose, this isolated information cannot be used to calculate the normalized amount of PPs formed per UV dose unit. Consequently, comparisons between quantitative studies are generally less than meaningful. Irradiation of spores with the full spectrum of sunlight at the Earths surface leads to the formation of SPDNA.51 However, quantitatively SPDNA formation is much higher on UV-C radiation: radiation at 254 nm is 103 times more efcient for SPDNA induction than that at 313 nm (UV-B) and 106 times more efcient than that at 365 nm (UV-A).51 Maximum SPDNA formation is reached near 260 nm.65 Because 254 nm monochromatic UV radiation accurately reproduces the effects of UV-C on DNA, the 254 nm wavelength emitted by low-pressure mercury lamps, also called germicidal lamps, is the one most commonly used in laboratory studies. Although in an early study no qualitative difference was observed in PP formation in the irradiated DNA spores, either dry or in an aqueous suspension,65 large variations in the quantitative yield of PPs have been recently reported (Table 1).63 When wild-type spores are irradiated in the dry state, PP formation appears to be dramatically lowered (15-fold) compared to irradiation in an aqueous suspension. Even though, as previously mentioned, experimental changes inherent in the different irradiation procedures may explainTable 1. Quantitative (Quant) and Qualitative (Qual) Yields of Intrastrand Bipyrimidine Photoproducts in Spores and Mutants Exposed to UV-C Radiation in Aqueous or Dry State Conditionsa ias-SPDNA 35/36 Quant Qual c,s T[CPD]T 5 Quant Qual sporeb wet dry wet dry wet dry 365 252 15.9 176 108 4.3 23.3 1.6 99.7 99.6 98.4 64.1 37.3 35.8 71.2 42.1 wild type 0.3 0.08 0.69 0.27 0.14 0.86 R/-type SASP 67 24.4 78.4 27 3.2 26.7 5.3 1.4 Ca-DPA Q 16.2 36.8 ND 0.10 0.07 Q 6 71.2 3.0 0.82 0.13 366.2 48 0.04 253.1 63 16.16 63 2.2 274.4 24.6 289.8 25 12.0 2.5 3.4 32.7 3.8 48 63 63 63 63 T[6-4]C 10 Quant Qual total refs

5. Factors Influencing SPDNA Formation in SporesAny comprehensive description of SPDNA formation must take into account its independent but complementary qualitative and quantitative aspects. From a qualitative standpoint, only the ratio (expressed in percent) of the total number of spore photoproducts vs the total number of PPs is considered. Therefore, the qualitative aspect represents only the formation ability of SPDNA compared to the other PPs. Conversely, the quantitative aspect represents the ability of the thymine residues to give rise to SPDNA. To quantify SPDNA formation, two methods are used. The rst method requires the use of DNA which has either 3H- or 14C-labeled thymine residues. After acid hydrolysis of SPDNA the results are expressed as the ratio (%) of the released SP versus the total number of thymine residues initially present in the DNA strand. The second method uses HPLC-mass spectrometry and standard solutions. It measures the amount of SPTIDE (for ias-SPDNA) and/or SPSIDE (for irs-SPDNA) released after enzymatic digestion of the SPDNA obtained for a particular UV dose range. This amount is then normalized to 104 bases, and the nal quantication, expressed as the number of PP per 104 bases per UV dose unit (J/cm2), can be calculated using the initial linear part of the curve, giving the number

R/-type SASP Q wet dry 4.0 0.31 1.9 8.9 Ca-DPA Q 78.0 37.6 1.2 34.3 82.1 1.3 39.6 207.4 37.1 3.5 63 63

a Expressed in lesions per 104 bases per J/cm2. b Terms indexed Q refer to the corresponding decient spore.

Spore Photoproduct


these observed quantitative yield variations, it is nonetheless clear that the irradiation state has little or no inuence on the respective distribution of PPs since both in the solid state and in suspension SPDNA remains the main PP generated in spore DNA. Since the aqueous environment has only a weak inuence on SPDNA formation, the impact of several other factors has been studied. Thus far, the most important factor is believed to be the association of spore DNA with specic spore proteins,namely,smallacid-solublesporeproteins(SASP).19,66

5.1. Small Acid-Soluble Spore ProteinsSASP are a group of spore proteins of low molecular weight (5-11 kDa, 60-75 residues). This group constitutes up to 10% of total spore proteins of dormant spores although it may comprise 20% of the spore core soluble proteins, a quantity sufcient to saturate the spore DNA. SASP, which are synthesized during sporulation, are rapidly degraded during germination and thereby provide the amino acids necessary for the bacterial protein synthesis. The SASP group is composed of different types of proteins: for B. subtilis the two principal ones are termed R/ and . For other species, the corresponding SASP are named R/- and -type SASP. Multiple R/-type SASP exist; they all have a very similar amino acid sequence and a molecular weight of 5-7 kDa. R/-Type SASP are monomers in solution. They are weak, nonspecic DNA-binding proteins and when bound to DNA form a dimer.67 In the spore this binding induces a strong modication of the UV-C DNA photoreactivity. In addition, R/-type SASP have a signicant effect on gene expression during sporulation and germination.68 A single -type SASP (molecular weight around 8-11 kDa) exists, and it does not appear to inuence the DNA photoreactivity. Its sole function appears to be supplying the necessary amino acids during the germination step. In R/-type SASP-less spore mutants, UV-C-induced SPDNA formation is quantitatively reduced by 50% compared to wild-type spores.69 Concomitantly, the formation of c,s T[CPD]T 5 undergoes a 40-fold quantitative increase.69 A subsequent and more accurate determination of the nature of the PPs formed by UV-C radiation together with their relative distribution has provided a better view of the impact of R/-type SASP on spore DNA photoreactivity.48,63 Even though R/-type SASP have no signicant inuence on the global amount of PP formed (i.e., their quantitative impact is negligible), they still dramatically drive the photochemistry of spore DNA toward ias-SPDNA formation (i.e., their qualitative impact is large). Indeed, in the presence of R/-type SASP, ias-SPDNA represents more than 99% of all the dipyrimidine PPs formed whereas in R/-type SASPless spore mutants ias-SPDNA represents only 37-64% of the dipyrimidine PPs.48,63 Interestingly, these studies also show that ias-SPDNA formation is not fully prevented in R/-type SASP-less spore mutants. Although R/-type SASP-less spores still contain up to 15% of R/-type SASP, this observation clearly indicates that, in vivo, factors other than SASP contribute to SPDNA formation. The amount of PPs formed in experiments using R/-type SASP-less spores is signicantly reduced when the irradiation is performed in the dry state.63 Linking R/-type SASP-modied DNA photoreactivity either to a DNA conformational change or to the consequence of an induced dehydrated state or to both has been proposed.

However, this question is still a matter of debate, and the precise reason for the observed modied DNA photoreactivity is still far from being fully understood. In living cells, DNA adopts a B conformation. The hypothesis of a different DNA conformation in spores was made as early as 1965.50 The early suggestion that spore DNA could adopt an A conformation was formulated 3 years later70 and then supported by in vitro experiments (IR and CD).71 Consequently, for several years spore DNA has been described as being in an A conformation, and this conformational switch has been held responsible for the specic photochemical behavior of spore DNA. However, electron microscopy studies have indicated that the base pair per helical turn in R/-type SASP-bound DNA is not signicantly different from that of vegetative cell DNA.72 This has led to the proposal of an A-like conformation for spore DNA,72,73 although more recent cryoelectron microscopy studies support a conformation close to the B form for spore DNA.74 A detailed picture of the structural interactions occurring between R/-type SASP and DNA has become recently available, nicely completing the few pieces of the puzzle that had been previously identied. R/-Type SASP bind only to double-stranded (ds) DNA (or ds ODNs).75 Although R/-type SASP exhibit a random coil conformation in the absence of DNA,76 upon binding to ds DNA, they become structured and hence R helical.76 Crystallographic resolution of a R/-type SASP bound to a 10 bp DNA duplex has revealed that the DNA helix adopts an A-like conformation with the base pair planes essentially parallel to each other and normal to the helix axis, an average value for the twist of 31.5, and all sugar puckering in the C3-endo conformation. However, because binding of R/-type SASP to DNA widens the minor groove, the rise per base pair of SASPbound DNA is now identical to that for B-DNA. Therefore, spore DNA adopts a A-B-DNA conformation.67 Binding to DNA facilitates SASP dimerization, is cooperative, and follows both local conformational changes of ds DNA around the bound R/-type SASP and also SASP-SASP interactions76 mediated by the N-terminal amino acid residues.77 Crystallographic observations have shown that when bound to DNA, each R/-type SASP monomer comprises two long helical segments, one lying on the edge of the DNA minor groove and the other located in the minor groove and connected by a turn region.67 The C-terminal residues are also implicated in DNA binding but so far only through unidentied interactions.78 Both N and C termini are devoid of secondary structure.67 R/-Type SASP binding to ds DNA is nonsequence specic even though it is modulated by the ds DNA sequence. Binding of R/-type SASP to DNA encompasses four75,76 to six base pairs79,67 and forms a helical coating around the DNA that greatly increases the DNA stiffness.72 Cryoelectron microscopy experiments have revealed that these helical laments (nucleoprotein helices) are tightly packed in a toroidal conformation by interdigitation of R/-type SASP domains from adjacent helices.74 Such assembly is stabilized by hydrophobic interactions and induces a substantial dehydration in the immediate vicinity of the DNA.74,67 This is believed to modify its photochemistry. Indeed, lack of water is known to change the DNA reactivity, and for example, the dehydrated state of the spore core is most likely responsible for the R/-type SASP protection of cytosine against hydrolytic deamination.80 The highly dehydrated state of R/-type SASP-bound DNA is currently believed to be the major factor responsible


Desnous et al.

for specic spore DNA photochemistry. This hypothesis is in good agreement with the observations made for isolated DNA81,82 (section 6.2.1). Nevertheless, the fundamental molecular and structural effects of the absence of water on the photoreactivity of DNA in spores remain unknown. An unidentied conformational change induced by dehydration or an unusual SASP-bound DNA rigidity cannot as yet be ruled out.

5.2. Dipicolinic AcidBecause SPDNA is formed in R/-type SASP-less mutants, R/-type SASP is clearly not the only factor governing SPDNA formation in spores. Among other factors inuencing SPDNA formation is dipicolinic acid, which is present in the spore as a Ca2+ chelate (39). Calcium dipicolinate constitutes 15% of the dry spore weight.83 It strongly absorbs in the UV-C at 271 and 278 nm and to a lesser extent at 263 nm.83,84

appear to be the most critical. Their cumulative absence in spore mutants results in a dramatic decrease of SPDNA formation and leads to spore DNA photochemical behavior qualitatively and quantitatively closely resembling that of vegetative cell DNA. When mutant spores lacking both R/type SASP and Ca-DPA are irradiated in the wet state, SPDNA is formed in only a 2% yield of the total dimeric PPs, whereas c,s T[CPD]T (5) and T[6-4]C (10) are formed in yields of 38% and 40%, respectively (Table 1).63 The decrease in the amount of PPs in the absence of both R/-type SASP and DPA is even more pronounced when spore mutants are irradiated in the dry state (Table 1).63

5.3. Pressure/Hydration LevelAmong the physical factors evaluated that are known to inuence SPDNA formation is that of pressure. This is because of the particular concerns regarding possible interplanetary contamination by spores. Under the Earths atmospheric pressure (101.3 kPa), the UV-C irradiation of spores leads almost exclusively to the formation of SPDNA. Under an ultrahigh vacuum of 2 10-6 Pa with a 254 nm UV dose of 0.5 J/cm2, SPDNA remains the main photoadduct (69%) but c,s T[CPD]T (5) and t,s T[CPD]T (40) are also formed in qualitative yields of 21% and 10%, respectively.65,90,91 Under ultrahigh vacuum, the quantitative formation of SPDNA is reduced by ca. 30% compared to that under atmospheric conditions.90 As observed in heat denaturation experiments, the partial DNA denaturation in the spore following the extreme dehydrated state induced by low pressure can explain the formation of t,s T[CPD]T 40.90,92 The photoreactivity of DNA in spores under a medium vacuum (1-2 Pa) is similar to that observed under ultrahigh vacuum.91

During sporulation, DPA is synthesized in the mother cell by DPA synthetase. This enzyme is composed of the two subunits spoVFA and spoVFB, also called DpaA and DpaB.85 DPA is then internalized into the spore core probably through proteins encoded by the spoVA operon and excreted in an early stage of spore germination.86 The role of Ca-DPA as a photosensitizer in spore SPDNA formation has been demonstrated using DPA-less spore mutants.87 In these mutants, depending on the experimental conditions, a 4-7-fold quantitative decrease of SPDNA formation is observed (Table 1).63 Additionally, although iasSPDNA remains the major PP produced (42-71%), its formation is less selective in these mutants than in wildtype spores. The concomitant formation of 5 is also observed (16-36%) (Table 1).63 The involvement of 39 as a photosensitizer in spores is in agreement with in vitro experiments performed with Ca-DPA.55,63,87 The participation of Ca-DPA in SPDNA formation may involve a selective triplet-state energy transfer from the UV-C-excited Ca-DPA to the thymine bases.63 Such selective energy transfer could explain the exclusive involvement of dithymine in SPDNA formation and consequently the absence of spore photoproducts involving cytosine residues.48,63 A full understanding of the mechanism of the involvement of Ca-DPA remains elusive. Full clarication would rst require a detailed examination of the excited-state properties of Ca-DPA. Interestingly, Ca-DPA also induces a decrease in the core hydration state.88 This has led to the suggestion that 39 may also act indirectly and participate with R/-type SASP in the highly dehydrated state of the spore core. In addition, Ca-DPA may bind, probably through intercalation to DNA. This could provide an additional hydration reduction mechanism in the immediate vicinity of the spore DNA.89 The involvement of 39 in the conformational modication of spore DNA has also been raised. However, for the present at least this hypothesis is not strongly supported and the role of Ca-DPA is considered to be chiey as a photosensitizer.63 In summary, among the chemical factors governing the spore DNA photoreactivity, R/-type SASP and Ca-DPA

5.4. TemperatureTemperature is the other physical parameter identied as inuencing SPDNA formation at dithymine sites in spores. Because high temperature causes DNA denaturation, only low-temperature effects can be studied. Quantitatively, the optimum temperature for SPDNA production is -80 C.93 At this temperature, SPDNA formation is twice that observed at 22 C and four times that observed at -196 C for a UV-C dose of 0.02 J/cm2. The combined effects of the humidity and temperature of the spore core on SPDNA formation have not yet been explored.

5.5. ConclusionsTwo physical (temperature and pressure) and three chemical (R/-type SASP, water, and Ca-DPA) factors have been clearly identied as being particularly critical for SPDNA

Spore Photoproduct


Figure 10. Representation of the inuence of chemical (green) and physical (pink) factors on SPDNA formation in bacterial spores.

formation. Although the exact elucidation at the molecular level of each step of the complex cascade of events linking these factors to SPDNA formation remains, dehydration, whether intrinsic or induced, of the spore DNA environment appears to be the most critical factor governing SPDNA formation. The different parameters identied so far are presented Figure 10.

Table 2. Quantitative (Quant) and Qualitative (Qual) Yields of Intrastrand Bipyrimidine Photoproducts in Isolated DNA Exposed to UV-C Radiation under Aqueous Conditions or in the Dry State in Both the Presence or the Absence of r,-Type SASP and Ca-DPAa ias-SPDNA 35/36 c,s T[CPD]T 5 T[6-4]C 10 Quant ND Qual Quant Qual Quant Qual total refs 187 15 27 10.1 7.4 2 0.2 10.42

6. Artificial Production of SPDNA, SPTIDE, and SPSIDEThe intriguing photochemical properties of the DNA present in spores have encouraged (bio)chemists to explore the specicity of SPDNA production and to dene the conditions permitting its formation outside the spores. Oligonucleotide models containing a denite number of spore photoproducts at known locations are valuable tools in understanding the SPDNA mechanism of formation for accurately studying its biological properties as well as for unraveling its repair processes.

solution 238 42.3 29.7 40.4 7.3 10.6 3.3 23.6 66.5 22.7 dry 111 45.1 9.72 32.3 7.8 37.1 R/-type SASP 49 46.2 5 11.5 DPA 30.4 46.34

33.3 562 20.4 73.5

48 96

18 3.19 0.7 solution dry drya

11 246.1 48 33.5 30.13 53 35.2 21 63 1.9 0.5 106 43.6 48 48 63

25 29 14.9

15.8 65.7

Expressed in lesions per 10 bases per J/cm .

6.1. UV Irradiation of Bacterial Vegetative CellsWhereas it has been well established that the UV-C irradiation of bacterial vegetative cells at room temperature does not lead to SPDNA, this latter lesion does form in the DNA of E. coli cells engineered to synthesize R/-type SASP (3% of total thymine for a UV dose of 2.5 J/cm2).94 When E. coli cells are exposed to UV-C irradiation at -79 C, SPDNA is also produced (1% of the total thymine for a UV dose of 0.2 J/cm2).95 This result conrms that neither R/type SASP nor Ca-DNA is absolutely necessary for SPDNA formation.

Table 3. Yields of SPDNA and c,s T[CPD]T 5 in Isolated DNA Produced by UV-C Irradiationa SPDNA, -100 C -99 C -100 C W/EG solution dry solution + R/-type SASP dry + R/-type SASP dry + R/-type SASP + Ca-DPAa

c,s T[CPD]T 5, 0.5 2 (+ U[CPD]T) 0.8 4.3 2.2 ND 0.7 BA-AA > EMA > BA). Mazurek et al.154 thoroughly described synthesis, morphologies, and mechanical properties of silicone bearing various terminal functions such as methacrylate, styrene, acrylamide, or methacrylamide copolymerized with various acrylate monomers by UV cure and led to similar conclusions as Yu et al.153 To sum up the macromonomer issue, it can be seen from this overview that silicone macromonomers with various functionalities have been available for a long time. It emerges that siloxane macromonomers are essentially obtained thanks to D3 ROP to reach monofunctional macromonomers. Besides, bifunctional macromonomers obtained by a simple one-step method allow preparing cross-linked polymeric materials. Both mono- and bifunctionalized PDMS are more and more extensively used thanks to their commercial availability.162

Scheme 29. Values of r2 vs Length of the Side Chain for the Copolymerization of AN (M2) with Various Methacrylatesa

a M1: MMA, BuMA, or methacrylic polysiloxane macromonomers PDMS-MA1 500 g mol-1 and PDMS-MA4 1110 g mol-1. Reprinted with permission from ref 166. Copyright 1966 Elsevier Ltd.

3.2.3. Macromonomers Reactivity in CopolymerizationIn general, the copolymerization parameters of a monomer and a macromonomer, r1 and r2, are assumed to be close when bearing a similar polymerizable group. This was the conclusion of Greber et al.,144 who determined in the

copolymerization of styrenic macromonomers (Scheme 19) with styrene reactivity ratios r1 r2 1 using the Mayo-Lewis method.163 Kawakami et al.145 also estimated that the reactivity ratios of both styrenic and methacrylic macromonomers were quite the same as those of the corresponding monomers but without measuring them accurately. Cameron and Chisholm149 determined the ratios r1 and r2 for the copolymerization of methacrylic polysiloxane macromonomers (M1) with styrene (M2). Despite the use of two different methods to reach these coefcients (FinemanRoss164 and Kelen-Tudos165), experimental errors prevented them from any meaningful values for macromonomer ratio r1. Nevertheless, a tendency was drawn: the value of r2 increases (from 1.06 to 1.55) as the molecular weight of the macromonomer increases (from about 500 to 10 000 g mol-1). According to the authors, this behavior is due to steric effects. In another study,166 under similar conditions except that the comonomer was acrylonitrile (AN), the authors reached the same conclusion as illustrated in Scheme 29. DeSimone et al.147,167,168 compared the copolymerization of methacrylic polysiloxane macromonomer with MMA by different polymerization processes: free radical, group transfer (GTP), and anionic polymerizations. They determined that the molecular weight distributions were logically narrower in GTP and anionic polymerization than in free radical polymerization. In addition, they nicely obtained the chemical composition distribution (CCD) using supercritical uid fractionation (SCFE) in supercritical chlorodiuoromethane, which is far less laborious and time consuming than other techniques such as solvent demixing fractionation169 or reversed-phase high-performance liquid chromatography.170 It appeared that CCD is much narrower in GTP and anionic polymerization than in free radical polymerization. Vinylsilane has been previously considered as a radically polymerizable monomer, although it rather behaves like a


Pouget et al.

transfer agent as discussed later in this review (section 3.3.1). Nevertheless, it is worth mentioning in this part that vinylsilane-terminated silicone macromonomers were found to copolymerize with VAc, to our knowledge the only monomer depicted in the literature able to copolymerize with vinyl silane.150152 Vinylsilanes otherwise do not homopolymerize but only form oligomers, which may be ascribed to the alpha silyl radical being too stable, i.e., unreactive toward the propagation step, thus only promoting transfer and/or termination reactions. Considering macromonomers, their low reactivity was additionally attributed to a macrophase separation during the polymerization process, since increasing the macromonomers feed did not generate chains with a higher graft density.

3.3. Polysiloxane Macrotransfer Agents3.3.1. Transfer to PDMSRadicals are known to react onto the methyl of the PDMS backbone (by hydrogen abstraction) from which polymerization can occur. Nevertheless, preparing copolymers this way is extremely challenging since the silylmethyl radicals thus formed can also recombine to yield a cross-linked material. Therefore, the resulting architectures are typically not well-dened copolymers but either grafted cross-linked silicone, when using common bulk and solution processes, or core-shell particles, when using a seeded emulsion polymerization process. Two main radical generator families are generally quoted in the literature. Nonvinyl-specic radical generators produce radicals onto a PDMS backbone, without the need for any reactive functions, to cross-link the materials. The most famous nonvinyl-specic vulcanizing agent is benzoyl peroxide. Vinyl-specic radical generators, such as di-tertbutyl peroxide, normally only react on PDMS functionalized with vinyl functions. Depending on the authors, this latter peroxide can proceed in different ways: either it is unable to generate a radical on a permethyl PDMS backbone171,172 or it generates a silylmethyl radical on a permethyl PDMS backbone, which however very quickly recombines with radicals derived from di-tert-butyl peroxide decomposition.173175 Whatever the type of radical generator used, PDMS cross-linking is induced by the reaction of radicals generated on the backbone of the silicone polymer, via a vinyl group or a methyl group, yielding interchain links, respectively composed of 3 or 2 methylene groups. Apart from this vulcanization process, it was shown recently that it is possible to use these generated radicals for copolymer synthesis thanks to a grafting from method. Vinylic siloxanes, cyclosiloxanes, or polysiloxanes exposed to a radical generator generally only lead to the formation of a dimer or more hardly to a trimer; we thus considered these vinyl-functionalized molecules as transfer agents rather than (co)polymerizable entities as often quoted in the literature. Some reports dealt with the radical copolymerization of vinylic monomers in the presence of vinylfunctionalized polysiloxane, but none of them focused on the characterization of the resulting copolymers. For example, toughened thermoplastics such as PMMA or PS were synthesized by polymerization initiated by radicals generated on the vinyl functions of PDMS.176 Dong et al.177 prepared PS-modied silicone elastomers in a similar manner. In a rst step, a vinyl containing hydroxyl-terminated polysiloxane was rst reacted with styrene and benzoyl peroxide, and

in a second step, cross-linking of the PDMS phase proceeded thanks to a trialkoxysilane. Using extraction techniques, the authors showed experimentally that soluble fractions were less important than the theoretical ones. They noticed that either formation of PDMS-g-PS during the rst step of styrene polymerization could occur or PS could be so entangled in the PDMS network that it could not be extracted. Analyses only focused on the nal cured blends, so that no information is available on the copolymer structures formed during the rst stage of the protocol. Indeed, whereas abstraction of hydrogen on vinyl-PDMS is possible, it has not been extensively studied in bulk or in solvent media. As in the case of transfer to nonvinylic polysiloxane, this may be due to the difculty encountered to control the resulting material architecture. A series of patents from the same assignee described rst the polymerization from a radical generated on a PDMS backbone and the generation of cross-linked materials from hydroxyl groups of the polydimethylsiloxane. Actually, as described in the examples, hydroxy-terminated PDMS was mixed with di-tert-butyl peroxide and various monomers such as styrene, butyl acrylate, or acrylonitrile. Afterward, the mixture was heated to lead to the corresponding grafted copolymers prior to cross-linking thanks to trichlorosilane or tri- or tetra-alkoxysilane and so forth.178,179 This was done in solvent media180 to obtain coatings by casting, in bulk in a reactor,181,182 or in extruders183 to give materials. A surprising fact is the use of di-tert-butyl peroxide, a vinylspecic radical generator, which is claimed to be preferred to benzoyl peroxide; the latter however falls in the scope of the invention. Enhanced mechanical properties such as tensile strength, elongation, and hardness were claimed, and coatings were more abrasion resistant. Thanks to this method, thixotropic polymers such as poly(dimethylsiloxane)-gpoly(methacrylic acid) were also obtained.184,185 To sum up this part, transfer to PDMS was not an intensive eld of research following the early studies on that topic in the 1970s, undoubtedly because of the difculty to control polymerization reactions especially with nonfunctionalized PDMS. This process however remains attractive in the latex eld thanks to the ease of process and its versatility as will be described in the following section.

3.3.2. Silicone Containing Core-Shell Particles via Radical PolymerizationCore-shell latex particles resulting from seeded emulsion polymerization are of high interest given their numerous applications such as coating modication, rubber strengthening, or thermoplastic and thermosetting polymer toughening. This last application generally involves a thermoplastic shell and an elastomeric core, the latter explaining the interest for the silicone eld. In direct emulsion, it is thermodynamically favored that the most hydrophobic polymer, in most cases PDMS, will form the core of the structure. However, some strategies have been developed to obtain core-shell structures where PDMS surrounds the particles. Note that most systems involve a polymerization of the shell around the core without any chemical links between them. Some of them will be considered as reference samples, but formation of a shell linked to the core through radical reactions is the purpose of this section. We rst discuss the formation of copolymers where unfunctionalized PDMS is used as the seed. Okaniwa and Ohta186,187 prepared PDMS-g-PS and PDMS-g-PAN copoly-

Poly(dimethylsiloxane)-Containing Copolymers

Chemical Reviews, 2010, Vol. 110, No. 3 1251 Scheme 30. Morphologies of Core-Shell Latexes Depending on the Nature of the PDMS Seed and the Miscibility of the Comonomers with PDMS

mers by forming a radical by hydrogen abstraction from the silylmethyl group of the silicone polymer in emulsion. The efciency of the grafting reaction was correlated to the initiator properties: its ability to generate an oxyradical, its oil solubility, and the absence of potentially abstractable protons in its structure which may consume the radical. Among the studied initiators, tert-butyl perlaurate reduced by FeSO4 led to the best grafting ratio. In addition, Okaniwa188 also investigated core-shell materials using PDMS/polybutadiene as the core and poly(styrene-co-acrylonitrile) as the shell. Polybutadiene is widely used for its elastomeric properties in the core-shell eld, and its grafting with thermoplastic polymers is relatively easy; its major drawback arises from its sensitivity to oxidation. Okaniwa overcame this disadvantage from the association of PB with PDMS which is especially oxidation resistant. The author showed that the modication of the PDMS with PB remained efcient (still using tert-butyl perlaurate/FeSO4 redox initiator). Thanks to such PB/PDMS seeds, Okaniwa could graft SAN more easily than on pure PDMS seeds. As already mentioned, initiation of polymerization from unfunctionalized silicone polymers is scarcely used because of the challenging control of the reaction. To the best of our knowledge, the rst introduction of vinyl-containing PDMS seed copolymerized with acrylic or styrenic monomers appeared in patents189191 which claimed improved thermal or mechanical properties. An increase of the vinyl content in the seed led to enhanced elongation at break and tensile strength of the resulting materials. He et al.192,193 studied more deeply core-shell particles with vinyl-PDMS as the starting material (resulting from the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8tetravinylcyclotetrasiloxane (D4V)). Actually, they compared two strategies in order to obtain core-shell materials. The vinyl-functionalized PDMS was either cross-linked before its use as a seed for the polymerization of the shell or directly used as the seed. In this latter case, cross-linking and polymerization occurred simultaneously. The authors observed that the formation of a core-shell structure was inuenced by (i) the reactivity ratios of the monomers and polysiloxane, (ii) the mobility of the species in the emulsion, (iii) the hydrophilicity of the monomers (the authors used a hydrosoluble initiator), and (iv) the compatibility of the vinyl monomers and their polymers with PDMS. For instance, using an emulsion polymerization with linear vinyl PDMS as the seed, they were able to prepare core-shell particles with BMA and MMA but not with styrene. By contrast, core-shell particles were obtained with cross-linked PDMS (i.e., in the absence of vinyl functions) with styrene but not with MMA. In a more complete study,194 the authors demonstrated that since styrene and poly(styrene) were compatible enough with the PDMS seed, they were able to penetrate the seed as long as PDMS was not cross-linked and consequently formed a nonsegregated copolymer latex. When PDMS is cross-linked, it acts as a shield and yields a PDMS-PS core-shell structure. On the contrary, MMA and PMMA are much less compatible with PDMS. Consequently, when the seed consisted of cross-linked PDMS, MMA was rejected and homopolymerized away from PDMS (Scheme 30). Kong and Ruckenstein195 prepared PDMS-poly(St-MMAAA) core-shell particles thanks to a similar seeded emulsion polymerization process. Five weight percent of D4V was added during the preparation of the PDMS seed by anionic

or cationic polymerization of D4. Using transmission electron microscopy (TEM), they observed that core-shell particles exhibited a more uniform particle size distribution than without D4V. 3-(Trimethoxysilyl)propyl methacrylate was also added to D4 during the polymerization to cross-link the seed whose diameter decreased. Unfortunately, its effect on the properties of the nal material was not extensively discussed. Analogous PDMS-PMMA or PDMS-PBuA core-shell particles using vinyl polysiloxane were obtained by Dai et al.,196 where polymerization was initiated by 60Co -ray irradiation. Films formed from these materials showed higher decomposition temperatures, enhanced pendulum hardnesses, lower water absorptions, but decreased tensile strengths as the PDMS concentration increased. Zou et al.197,198 observed that the particle size greatly inuences the lm properties such as mechanical performance, water absorption, and transparency. For instance, increasing the vinyl content from 1.1 10-3 to 2.2 10-3 mol per gram of seed emulsion led to a decrease of particle size and a 20% enhancement of the tensile strength of a PDMSP(MMA-co-BA) lm. On the other hand, the evolution of the elongation at break was negligible. Lin et al.199 also prepared PDMS-poly((meth)acrylate) (MMA and BuA) core-shell particles, but they observed much more complex morphologies when 10 wt % of D4V were added to D4 during ROP of the PDMS seed. Actually, this led to a multiglobular shell surrounding a shapeless PDMS core. As demonstrated by Soxhlet extraction of the particles, the structure was indebted to cross-linking occurring by copolymerization of acrylate monomers with vinyl groups. Contact angle measurements showed the more hydrophobic character of the lm resulting from the D4V-containing seeds, giving evidence for a partial shell formation. In order to obtain a core-shell structure with PDMS as the shell, Kong et al.200 prepared rst a cross-linked PMMA seed using ethylene glycol dimethacrylate (EGDMA) and then a D4V/ammonium persulfate mixture was added at the end of the MMA polymerization. D4V vinyl functions were supposed to react with residual MMA of the PMMA seed in formation. Afterward, the shell was obtained by polymerizing D4. The authors compared a lm made from the obtained latex with one formed from a similar core-shell latex which did not involve D4V. TEM observations showed that PDMS containing vinyl groups formed a continuous phase in the rst lm (in presence of D4V). Consequently,

1252 Chemical Reviews, 2010, Vol. 110, No. 3 Scheme 31. Chemical Initiation Mechanism of Telomerizationa

Pouget et al.

3.3.3. Chemical-Initiated TelomerizationThe telomerization process consists of reacting, under polymerization conditions, a molecule YZ, which is called a telogen, with more than one unit of a compound M containing an unsaturated group, called a taxogen, to form products called telomers Y(M)nZ.204,205 The general mechanism of telomerization by chemical initiation is given in Scheme 31. By functionalizing a PDMS chain, it is possible to transform it into a macrotelogen. Telomerization differs from polymerization in the following issues: the fragments of the initiator mainly induce the rupture of the telogen, whereas in polymerization they add onto the monomer; the number of M units in the nal product is larger than 1 but lower than 100; the functional groups on both chain ends can be easily transformed thanks to the low molecular weights of the resulting polymers. Several authors carried out the synthesis of block or graft copolymers using a PDMS macrotelogen, as summarized in Table 3. They all carry a thiol function except the one used by Tezuka that bears a Si-H functionality (Table 3, entry 5).206 The rst synthesis of PDMS graft copolymers using telomerization has been carried out by the Dow Corning Corp.207 in 1969 using two different emulsion pathways. In the rst step, siloxanes 1 (Table 3, entry 1) were prepared by emulsion copolymerization of the corresponding trimethoxysilane as described by Hyde208 and Findlay.209 The second step is the addition of the vinyl monomer and ammonium persulfate to the nitrogen-purged emulsion to perform the polymerization by heating. In a second approach, the polysiloxane that has been previously prepared by bulk

a YZ is the telogen, and M is an unsaturated monomer that can react by radical polymerization.

its hardness was lower than the reference sample without D4V. It should be noticed that 3-methacryloxypropyl trimethoxysilane (MATS) was also used to prepare such core-shell architectures. This molecule can copolymerize with MMA201,202 or styrene203 when added in the last moments of the seed polymerization. Afterward, methoxysilane groups reacted with D4 to give the core-shell structure. To summarize this core-shell section, radical polymerization allowed researchers to obtain small and ne particles with well-dened morphologies, which is a key issue for the properties of the resulting materials.

Table 3. Telogens and Monomers Used To Make Block and Graft PDMS-Based Copolymers by Telomerization


Chemical Reviews, 2010, Vol. 110, No. 3 1253 Scheme 32. General Scheme of Reversible ActivationDeactivation in Controlled Radical Polymerization217

or solution techniques is emulsied, the second step being identical to the one described above. Falender et al.22,23,210,211 from the Dow Corning Corp. also performed the telomerization of many different monomers (acrylic, methacrylic, styrenic monomers) using a PDMS macrotelogen (Table 3, entry 2). For example, they carried out the synthesis of PDMS-g-poly(styrene-co-acrylonitrile) of high impact strength using a mercaptopropyl PDMS as a telogen in water suspension or in bulk with benzoyl peroxide as radical initiator. Saam and Tsai212 carried out the dispersion polymerization of methyl methacrylate in aliphatic hydrocarbons containing poly(dimethylsiloxane) modied with mercaptoalkyl side groups (Table 3, entry 2). They observed the formation of particles stabilized by a protective layer of solvated poly(dimethylsiloxane). The evolution of particle size was studied while polymerizing MMA. In the very early stage of polymerization, a large number of very small particles of broad size distribution was formed. These particles contained a relatively high amount of silicone stabilizer in the form of graft copolymers. As the conversion increased, the particles tended to agglomerate to give some larger particles and a broad size distribution. Between 5% and 10% conversion, a critical point was reached: the smaller particles were absorbed by the larger particles present in the system. From this point, new particles were no longer formed (end of nucleation) and polymerization occurred within the swollen monomer particles. This was evidenced by a steady particle growth and a nal narrow size distribution. Our group also performed the synthesis of triblock cooligomers of poly(methyl methacrylate)-b-PDMS-b-poly(methyl methacrylate)213 by telomerization. The rst step consisted of the synthesis of a macromonomer containing a PMMA chain using -mercaptopropylmethyldimethoxysilane as telogen (Table 3, entry 3), MMA as monomer, and AIBN as initiator. The second step was the condensation reaction between the macromonomer and the silanol-terminated PDMS at 100 C for 20 h in the presence of a catalyst (2-ethylhexanoic acid/tetramethylguanidine complex). The presence of small amounts of diadduct (reaction of the second methoxy silyl group with a silanol-terminated PDMS) has been observed. More recently, Fawcett et al.214 carried out the telomerization of different monomers (styrene, MMA, chloroprene) (Table 3, entry 4) using mercaptopropyl-functionalized PDMS and AIBN as radical initiator. The authors observed that the thiol/monomer ratio controls the reaction type: for a low monomer/macrotelogen ratio, only one monomer unit reacted with the SH group (thiol-ene reaction), whereas a higher concentration ratio resulted in graft and block copolymers. Thermal-initiated telomerization was also carried out in scCO2 to prepare PMMA particles.215 Okubo and co-workers used a triblock copolymer PMMA-b-PDMS-b-PMMA as colloidal stabilizer, prepared in situ by telomerization using a mercaptopropyl telechelic PDMS as telogen with AIBN as initiator (Table 3, entry 4). The particle diameter could be controlled by the concentration of the telogen which serves as steric stabilizer, with sizes ranging from submicrometers to micrometers. A very small consumption of telogen was observed, albeit producing sufcient copolymers to stabilize the PMMA particles. Tezuka et al.206 used telogen 5 (Table 3, entry 5) to prepare poly(vinyl alcohol)-b-PDMS-b-poly(vinyl alcohol) (PVAb-PDMS-b-PVA) block copolymers. The rst step was the telomerization of VAc in the presence of dimethylchlorosi-

lane as telogen and AIBN as radical initiator in benzene. The second step was the coupling reaction of the chlorosilaneterminated poly(vinyl acetate) (PVAc) prepolymer with a R,telechelic silanolate-terminated PDMS. Finally, PVA-b-PDMSb-PVA block copolymers were obtained by saponication of the PVAc-b-PDMS-b-PVAc block copolymer using a 10% aqueous sodium hydroxide solution in methanol. However, due to the sensitivity of PDMS to basic conditions, a loss of more than 15 wt % of siloxane was observed during this last hydrolysis step. The literature already largely described telomerization of a wide range of monomers.204,205 To date, mainly thiol-type PDMS were used as telogens. Using PDMS as a macrotelogen is an easy way to prepare silicone-containing copolymers, keeping in mind that the nal product does not exhibit high molecular weights. Moreover, chemical-initiated telomerization is compatible with dispersed aqueous or scCO2 media (suspension, emulsion, dispersion polymerizations).

4. Copolymers Obtained by Controlled Radical PolymerizationThe controlled radical polymerization (CRP)216 process includes a group of radical polymerization techniques that have attracted much attention over the past decade since they provide simple and robust routes to the synthesis of welldened polymers, low-dispersity polymers, and preparation of novel functional materials. The general principle of the reported methods relies on a reversible activation-deactivation process between dormant chains (or capped chains) and active chains (or propagating radicals) with different rate constants kact and kdeact, respectively (Scheme 32).217,218 The past few years have witnessed rapid growth in the development and understanding of new CRP methods. The most popular CRP methods treated in the following text are iniferters,219 nitroxide-mediated polymerization (NMP)220 that requires alkoxyamines, atom-transfer radical polymerization (ATRP)221 involving alkyl halides, metallic salts, and ligands, iodine-transfer polymerization (ITP) using alkyl iodides,141 and reversible addition-fragmentation chain transfer polymerization (RAFT)222 using dithiocarbonyl derivatives.

4.1. IniferterIniferters219 are specic agents that proceed in a radical polymerization via initiation, propagation, primary radical termination, and transfer to initiator (Scheme 33). Because bimolecular termination and other transfer reactions no longer dominate the polymerization, these polymerizations are performed by insertion of the monomer molecules into the iniferter bond, thus generating polymer chains with two initiator fragments at the chain ends. These thermal iniferters give controlled molecular weights but do not present a living chain end (i.e., no possibility of further chain extension). PDMS-based macroiniferters are given in Table 4 (entries 1 and 2).

1254 Chemical Reviews, 2010, Vol. 110, No. 3 Scheme 33. Main Steps Involved in the Mechanism of Iniferter Polymerization

Pouget et al.

Table 4. PDMS Thermal- and Photo- Macroiniferters

Macroiniferter 1 (Table 4, entry 1) was synthesized by a hydrosilylation reaction between Si-H-terminated PDMS and allyl-N-methylamine. Subsequent chain extension of the macrodiamine by the thiocarbamylation reaction using CS2 and oxidative coupling using I2 led to the formation of the polymeric iniferters (Scheme 34). Macroiniferter 1, when used under thermal initiation in the presence of vinylic monomers, gives a triblock copolymer with a central vinylic polymer block and a PDMS segment

at both ends. This macroiniferter was used for styrene and MMA polymerization between 60 and 100 C in bulk or in toluene solution.223,224 When the polymerization of styrene or MMA was carried out in concentrated solutions or in bulk, a slight microsegregation was observed due to the incompatibility of the PDMS segment with the propagating polymer chain. This polymer demixtion led to a decrease of the macroiniferter efciency.223 Moreover, the authors observed a decrease in chain transfer with increasing PDMS chain

Poly(dimethylsiloxane)-Containing Copolymers Scheme 34. Synthesis of Macroiniferter 1 (Table 4, entry 1)


length (Mn ) 2800-4200 g mol-1), which also suggested the occurrence of some phase segregation.224 The azeotropic temperatures of the iniferter, where ktr ) kp or Ctr ) ktr/kp ) 1, were determined to be 125 C for MMA and 110 C for styrene. When Ctr ) 1, the molecular weight remains constant during the polymerization. When the temperature is lower than the azeotropic temperature, the polymerization is not controlled and Mn decreases as the conversion proceeds (i.e. Ctr < 1). The authors found that for this macroiniferter Ctr(styrene) ) 1.5 Ctr(MMA). Macroiniferter 2 (Table 4, entry 2) is the equivalent of macroiniferter 1 but starting from a difunctionnal PDMS chain. When a vinylic monomer is polymerized in the presence of this particular transfer agent, a multiblock copolymer is formed. In the polymerization of MMA, Nair et al.228 showed that when the concentration of the macroiniferter increased, a retardation effect due to the participation of thiuramyl radicals in termination reactions was observed.

The molecular weight increased linearly with conversion, and the nal PDIs of the multiblocks were around 2-2.5. These authors thus reported the successful synthesis of PSPDMS and PMMA-PDMS multiblock copolymers, whereas when polymerizing acrylamide to obtain amphiphilic copolymers, a strong phase segregation occurred and led to an insoluble material due to some physical cross-linking. Photoiniferters are iniferters that are activated under UV radiation. These iniferters have the advantage that they lead to living chains. Indeed, the polymer chain resulting from termination can be reactivated by cleaving the C-S bond under UV radiation (Scheme 35). The different PDMS-based macrophotoiniferters reported here are quoted in Table 4 (entries 3-5). The rst study on photoinitiated polymerization was carried out by Inoue et al.43 They modied the surface of a cross-linked poly(dimethylsiloxane-co-methyl chloromethylsiloxane) by photopolymerizing hydrophilic monomers

Scheme 35. Main Steps Involved in the Mechanism of Photoiniferter Polymerization

1256 Chemical Reviews, 2010, Vol. 110, No. 3 Scheme 36. Synthesis of the PDMS Photoiniferter and Grafted Polymer Obtained

Pouget et al.

Scheme 37. General Mechanism of Nitroxide-Mediated Polymerization

(Table 4, entry 3) using diethyldithiocarbamated PDMS as photoiniferter. The synthesis of this photoiniferter and the grafted polymer obtained is described in Scheme 36. By this method, they successfully prepared hydrophilic surfaces which could nd applications in biomedical devices. Macrophotoiniferter 4 (Table 4, entry 4) was successfully used for the controlled polymerization of MMA and copolymerization of methyl acrylate and acrylic acid under UV radiation.230 Moreover, the living character of the chain ends was demonstrated by further growing two blocks of poly(2hydroxyethylacrylate) (PHEA) from the PMMA-b-PDMSb-PMMA triblock copolymer. Finally, VAc and 1,1dihydroperuorooctyl acrylate (FOA) were successfully polymerized by macrophotoiniferter 5 (Table 4, entry 5) under UV radiation.232 In the case of VAc, diblock copolymers with PDI ) 1.4 (at 30% monomer conversion) were obtained. Until now, PDMS-based macroiniferters were used for the controlled polymerization of styrene, MMA, methyl acrylate (MeA), acrylamide, AA, MAA, HEMA, HEA, AMPS, DMAEMA, sodium styrene sulfonate (NaSS), N-vinyl pyrrolidone (NVP), and FOA. Iniferters allow the controlled polymerization of a wide range of monomers,219 but the control over the molecular weights is poorer than by other PRC methods and photoactivation is required to achieve living character.

4.2. NMP: Nitroxide-Mediated PolymerizationIn nitroxide-mediated polymerization (Scheme 37),220 the reversible termination is the key step to keep a low concentration of propagating radicals in the reaction medium. All polymer chains should only be initiated by the alkoxyamine, leading to well-controlled polymer architectures. The nature of the counter radical is essential for the controlled character of the polymerization. The alkoxyamine has a rather labile C-O bond which cleaves homolytically upon heating to liberate the radical on the polymer chain, which propagates, and the counter radical, which is only able to end cap the propagating chains. The equilibrium is overwhelmingly shifted to the left (dormant chains), and the control is ensured thanks to the

persistent radical effect (i.e., accumulation of nitroxides).233,234 Table 5 summarizes the different precursors used in the synthesis of PDMS-based copolymers by NMP techniques. Styrene has been the most widely studied monomer for nitroxide-mediated polymerization from a PDMS precursor. As for conventional free radical polymerization, azo macroinitiators can be used to initiate the polymerization. Yoshida et al.235,236 used a bicomponent system consisting of a multiblock azo macroinitiator 1 (Table 5, entry 1) (Mn ) 30 800 g mol-1 with PDMS segments Mn ) 5000 g mol-1) and 4-methoxy2,2,6,6-tetramethylpiperidine-1-oxyl (MTEMPO) (Scheme 38). As soon as the azo macroinitiator decomposed, the formed radicals were trapped by MTEMPO to create the active macroalkoxyamine in situ. Styrene was polymerized in bulk at 130 C for 72 h. Due to the low initiator efciency, the resulting copolymer exhibited an AB diblock structure rather than the expected ABA triblock one. A chain extension with p-methoxystyrene showed the living character of the polymer chain end. Macroinitiator 1 (Table 5, entry 1) was also used by Ryan et al.237 in the dispersion polymerization of styrene in scCO2 using SG1 (N-tert-butyl-N-(1-diethylphosphono-2,2-dimethylpropyl)nitroxide) (Scheme 38) as a control agent at 110 C. A rather broad bimodal molecular weight distribution (MWD) was obtained. The use of AIBN as co-initiator and the reduction of the number of azo moieties in the azo macroinitiator tended to reduce the bimodality while introducing sufcient siloxane units to stabilize the dispersion in scCO2. The azo moieties were partially consumed by heating the macroazo initiator before adding the monomer to reach a new precursor with Mn) 21 800 g mol-1 and only 25% remaining azo moieties. A chain extension showed the high livingness of the polymer chains. The broad MWD was ascribed to polysiloxane chains bearing SG1 moieties at one or two chain ends, leading to di- or triblock copolymers, respectively. When a bicomponent system is used (azo macroinitiator + nitroxide), the low initiator efciency leads to an illdened architecture and a mixture of homopolymers and diblock and triblock copolymers.220 To obtain a well-dened di- or triblock copolymer, it is better to use a monocomponent system, assuring a better controlled architecture. Therefore, several organolithium alkoxyamines were used as initiators for the anionic polymerization of D3, leading to PDMS alkoxyamines. These macroalkoxyamines were further used for the nitroxide-mediated polymerization of styrene, leading to well-dened diblock copolymers. This method was rst applied by using macroalkoxyamine 2 (Table 5, entry 2 and Scheme 39) for the bulk polymerization of styrene238 at 120 C. The controlled polymerization proceeded up to 20% conversion, giving the desired diblock copolymer. However, above 20% styrene conversion, SEC analysis showed a bimodality, indicating the formation of homopoly(styrene) along with low molar mass diblock copolymer. Macroalkoxyamine 3 (Table 5, entry 3) was used to polymerize styrene in bulk at 120 C.239 The macroalkoxyamine reacted quantitatively to form diblock copolymers, and a linear evolution of molecular weight with conversion proved the controlled character of the polymerization. A nal PDI of 1.3 above 25% conversion was obtained. However, maximum monomer conversion was limited to 42%. Macroalkoxyamine 4 (Table 5, entry 4) was used for bulk and solution polymerizations of styrene240 at 120 C where some discrepancies between the molecular weights determined by SEC and NMR were observed. Moreover, the



Table 5. PDMS Macroalkoxyamines and PDMS Macroinitiators Used in Nitroxide-Mediated Polymerization

Scheme 38. Formulae of TEMPO, MTEMPO, and SG1

resulting copolymer did not have the expected composition from the monomer feed. Macroalkoxyamine 5 (Table 5, entry 5) was synthesized by hydrosilylation,241,242 thus avoiding the presence of the rather fragile Si-O-C bond.78,79 This macroalkoxyamine was usedScheme 39. Synthesis of Macroalkoxyamine 2 (Table 5, entry 2)

for the solution polymerization of styrene,241 leading to a mixture of homopoly(dimethylsiloxane), homopoly(styrene), and diblock copolymer. The presence of homoPDMS proved the incomplete reaction of the macroinitiator, while homoPS resulted from some thermal autoinitiation. However, by optimizing the reaction conditions, the amount of homopolymers was reduced, leading to a practically pure diblock copolymer.242 Nevertheless, some inhomogeneity in SEC could be attributed to diblocks with varying PS and PDMS content. Moreover, NMR analysis only showed around 30% of remaining TEMPO (2,2,6,6-tetramethylpiperidinyl-1-oxy) units on the chain ends. The difference was attributed to some hydrogen transfer,220,244 leading to -CHdCH-Ph and -CH2-CH2-Ph chain ends.


Pouget et al. Scheme 40. Mechanism of Atom-Transfer Radical Polymerization

Because TEMPO has no ability to control the radical polymerization of vinyl monomers other than styrene and styrene derivatives, Miura et al.243 used macroalkoxyamine 6 (Table 5, entry 6) to polymerize styrene and other monomers. The TIPNO (N-tert-butyl 1-phenyl-2-methylpropylaminoxyl)-based alkoxyamine has been reported to act as an excellent mediator for a wide range of vinyl monomers including styrene. Styrene was polymerized in bulk at 120 C. A linear evolution of the molecular weight with conversion was observed, and SEC showed a monomodal MWD with a PDI of 1.2, a value lower than those obtained at similar conversion with TEMPO-bearing control agents.238,239 TIPNO, like SG1, is a better control agent than the commercially available TEMPO. The TIPNO-based alkoxyamine 6 (Mn ) 4000 g mol-1, PDI ) 1.09) was further used for the polymerization of methyl acrylate (MeA) in bulk at 120 C.243 A linear evolution of molecular weights with conversion was observed with a nal PDI value below 1.2 at 50% conversion. A good correlation between experimental and theoretical molecular weights was observed. Moreover, to show the living character, the PDMS-b-poly(MeA) diblock copolymer was further used as a macroalkoxyamine in the polymerization of styrene, leading to an ABC triblock copolymer with PDI ) 1.4. Alkoxyamine 6 was also used for the controlled polymerization of isoprene (IP) in bulk at 120 C.243 The nal diblock copolymer presented a PDI of 1.15 and a good agreement was noticed between experimental and theoretical molecular weights. The diblock copolymer was further used in the polymerization of styrene to synthesize a PDMS-b-poly(IP)-b-PS triblock copolymer with a monomodal MWD (PDI ) 1.37). To date, NMP from PDMS macroalkoxyamines was essentially used for styrene polymerization. Only Miura et al.243 polymerized methyl acrylate and isoprene from a PDMS macroalkoxyamine. It is known that nitroxidemediated polymerization allows the controlled polymerization of a wide range of monomers.220 Among others, the controlled polymerization of acrylates (such as tert-butyl acrylate (tBuA), nBuA, or MeA), styrenics (such as styrene, p-bromostyrene, p-chlorostyrene, p-epoxystyrene, p-methoxystyrene, p-acetoxystyrene, p-chloromethylstyrene, p-tertbutoxy styrene, or styrene p-sulfonic acid sodium salt), isoprene, 4-vinyl pyridine, acrylonitrile, maleic anhydride, isopropylacrylamide, and N,N-dimethylacrylamide could be considered in the future. Indeed, all these monomers can be potentially polymerized using nitroxide-mediated polymerization from a poly(dimethylsiloxane) macroalkoxyamine, giving a wide range of different properties to the second block, which could lead to a variety of new applications. Furthermore, NMP of PDMS macromonomers has not been reported yet to yield graft copolymers. This is probably due to the limited range of commercially available PDMS macromonomers, since those based on methacrylates could not be easily controlled by NMP. Recent progress in NMP of methacrylates now makes it possible.245,246

control and the lower the extent of irreversible termination. Irreversible termination occurs as in conventional free radical polymerization by combination or disproportionation. Several reviews from Matyjaszewski247251 treat of the synthesis of organic-inorganic hybrid materials using atomtransfer radical polymerization. They all deal with the general synthesis routes used to obtain such hybrid materials, but none of them gives an extensive overview of the existing state of the art. Note that a patent from Matyjaszewski et al. covers

Chemical Reviews - March 2010

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