DETERMINATION OF POLYMER STRUCTURES, SEQUENCES, AND

ARCHITECTURES BY MULTIDIMENSIONAL MASS SPECTROMETRY

A Dissertation

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Doctor of Philosophy

Aleer Manyuon Yol

August, 2013

DETERMINATION OF POLYMER STRUCTURES, SEQUENCES, AND

ARCHITECTURES BY MULTIDIMENSIONAL MASS SPECTROMETRY

Aleer Manyuon Yol

Dissertation

Approved: Accepted:

______Advisor Department Chair Dr. Chrys Wesdemiotis Dr. Michael J. Taschner

______Committee Member Dean of the College Dr. Leah P. Shriver Dr. Chand K. Midha

______Committee Member Dean of the Graduate School Dr. Claire A. Tessier Dr. George R. Newkome

______Committee Member Date Dr. Wiley J. Youngs

______Committee Member Dr. Yu Zhu

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ABSTRACT

The matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry (MALDI-ToF/ToF MS) characteristics of different polystyrenes and polybutadienes are discussed in this dissertation. The compounds examined include linear, cyclic, in-chain substituted, and star-branched polymers as well as copolymers of styrene and either para-dimethylsilyl styrene (p-DMSS) or meta-dimethylsilyl styrene

(m-DMSS).

Chapter IV describes the differentiation of cyclic and linear polymers by 2D- mass spectrometry. The silverated quasimolecular ions from cyclic and linear polystyrenes and polybutadienes, formed by MALDI, give rise to significantly different fragmentation patterns in tandem mass spectrometry (MS2) experiments. With both architectures, fragmentation starts with homolytic cleavage at the weakest bond, usually a C–C bond, to generate two radicals. From linear structures, the separated radicals depolymerize extensively by monomer losses and backbiting rearrangements, leading to low-mass radical ions and much less abundant medium- and high-mass closed-shell fragments that contain one of the original end groups, along with internal fragments. With cyclic structures, depolymerization is less efficient, as it can readily be terminated by intramolecular H-atom transfer between the still interconnected radical sites

(disproportionation). These differences in fragmentation reactivity result in substantially different fragment ion distributions in the MS2 spectra. Simple inspection of the relative

iii intensities of low- vs. high-mass fragments permits conclusive determination of the macromolecular architecture, while full spectral interpretation reveals the individual end groups of the linear polymers or the identity of the linker used to form the cyclic polymer.

Chapter V presents the first sequence analysis of styrenic copolymers by tandem MS.

Copolymers of para-dimethylsilyl styrene (p-DMSS) or m-DMSS with styrene were prepared by living anionic polymerization. The MALDI-MS2 results for p-DMSS indicate that a block copolymer is formed, with the para-substituted styrene incorporated near the initiator. On the other hand, the MS2 results of m-DMSS reveal that a random copolymer is formed, consistent with comparable reactivities for m-DMSS and styrene.

These findings suggest that p-DMSS is more reactive than m-DMSS. The single-stage

(1D) MALDI-MS results further show that linear and 2-armed architectures are formed with both the m-DMSS and the p-DMSS comonomers.

The last Chapter, VI, focuses on the differentiation of linear in-chain substituted, cyclic, and star-branched polystyrene (PS) by tandem mass spectrometry. The in-chain functionalized PS gives a MS2 fragmentation pattern that is different from the one observed for cyclic PS with two linker units and, again, with a simple inspection of the tandem mass spectra, these architectures can easily be distinguished. The four-arm star- branched polymer investigated mainly breaks down by losing arms under MALDI-MS2 conditions.

Overall, this dissertation documents the usefulness of combined 1D and 2D mass spectrometry experiments for the identification of polymer substituents and their location, for distinguishing polymer architectures, and for determining copolymer sequences.

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The results presented in this dissertation have been published or are pending for publication in the following journals.

1. Quirk, R. P.; Wang, S-F.; Foster, M. D.; Wesdemiotis, C.; Yol, A. M. “Synthesis of Cyclic Polystyrenes Using Living Anionic Polymerization and Metathesis Ring-Closure” Macromolecules 2011, 44, 7538-7545.

2. Liu, B.; Quirk, R. P.; Wesdemiotis, C.; Yol, A. M.; Foster, M. D. “Precision Synthesis of ω-Branch, End-Functionalized Comb Polystyrenes Using Living Anionic Polymerization and Thiol-Ene “Click” Chemistry” Macromolecules 2012, 45, 9233-9242.

3. Yol, A. M.; Dabney, D. E.; Wang, S-F.; Laurent, B. A.; Foster, M. D.; Quirk, R. P.; Grayson, S. M.; Wesdemiotis, C. “Differentiation of Linear and Cyclic Polymer Architectures by MALDI Tandem Mass Spectrometry (MALDI-MS2)” J. Am. Soc. Mass Spectrom. 2013, 24, 74-82.

4. Quirk, R.P.; Chavan, V.; Janoski, J.; Yol, A.; Wesdemiotis, C. “General Functionalization Method for Synthesis of α-Functionalized Polymers by Combination of Anionic Polymerization and Hydrosilation Chemistry” Macromolecular Symposia 2013, 323, 51-57.

5. Yol, A. M.; Janoski, J.; Quirk, R. P.; Wesdemiotis, C. “Sequence Analysis of Styrenic Copolymers by Tandem Mass Spectrometry” Anal. Chem. (Submitted)

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DEDICATION

To the memory of my father and mother.

To all my brothers, sisters, nephews, nieces, brothers-in-law, and sisters-in-law.

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TABLE OF CONTENTS

Page

LIST OF TABLES………………………………………………………...... x

LIST OF FIGURES………………………………………………………………………xi

LIST OF SCHEMES……………………………………………………………………xvi

CHAPTER

I. INTRODUCTION………………………………………………………………...1

II. MASS SPECTROMETRY BACKGROUND…………………………………….5

2.1. Ionization techniques…………………………………………………………5

2.2. MALDI……………………………………………………………………….5

2.3. Mass analyzers………………………………………………………………..8

2.3.1 Mass resolution……………………………………………………..9

2.3.2 Time of fight (ToF) mass analyzer………………………………...10

2.3.2.1. Reflectron………………………………………………..12

2.3.2.2. Delayed extraction (DE) or pulsed ion extraction (PIE) or time lag focusing……………………………………………...... 13

2.4. Detectors…………………………………………………………………….15

2.4.1. Microchannel plate (MCP) detector……………………………….15

III. MATERIALS AND EXPERIMENTAL PROCEDURES………………………17

3.1. Materials…………………………………………………………………….17

3.1.1. Linear and cyclic polymers…...... 17

3.1.2. Polystyrene copolymers………………...…………………………20

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3.1.3. Linear in-chain functionalized precursor, cyclic with two linker units, and four star polystyrenes………………...... 20

3.2. MALDI experimental procedures………………………………………...…21

3.3. MALDI instrumentation…...... 22

IV. DIFFERENTIATION OF LINEAR AND CYCLIC POLYMER ARCHITECTURES BY MALDI TANDEM MASS SPECTROMETRY (MALDI-MS2)…………………………………………………………………...24

4.1. Linear Polystyrene…………………………………………………………..24

4.2. Cyclic Polystyrenes…………………………………………………………30

4.3. Cyclic Polybutadiene……………………………………………………...... 43

4.4. Conclusions…………………………………………………………….…....48

V. SEQUENCE ANALYSIS OF STYRENIC COPOLYMERS BY TANDEM MASS SPECTROMETRY………………………………………………………49 5.1. Composition and architecture of poly(dimethylsilylstyrene-co-styrene) copolymers ………………………………………………………………………49

5.2. Reference MS2 spectra of polystyrene and poly(p-DMSS-b-styrene)………54 5.3. Sequence analysis of poly(p-DMSS-co-styrene) and poly(m-DMSS-co- styrene)…………………………………………………………………………...61 5.4. Conclusions…...... 68

VI. MALDI-TOF/TOF TANDEM MASS SPECTROMETRY OF LINEAR IN- CHAIN SUBSTITUTED, CYCLIC WITH TWO LINKER UNITS, AND FOUR- ARM STAR-BRANCHED POLYSTYRENES……….………………………...69

6.1. Linear in-chain substituted PS…………………………………………...….69

6.2. Cyclic PS with two linker units……………………………………………..74

6.3. 4-arm star-branched polystyrene…………………………………………….79

6.4. Conclusions………………………………………………………………….87

VII. SUMMARY……………………………………………………………………...88

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REFERENCES…………………………………………………………………..90

APPENDIX………………………………………………………………………98

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LIST OF TABLES

Table Page

2.1. Common MALDI matrices…………………………………………………………..7

2.2. Common lasers used for MALDI experiment………………………………………..8

5.1. Measured vs. calculated monoisotopic m/z values of the oligomers observed in the low (m/z 1490-1630) and high (m/z 3530-3710) mass regions (Figures 5.2b-c) of the MALDI mass spectrum of poly(p-dimethylsilylstyrene-co-styrene).……………………53

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LIST OF FIGURES

Figure Page

2.1. MALDI principle…………………………………………………………………...... 6

2.2. Resolving power…………………………………………………………………….10

2.3. Linear time of fight principle………………………………………………………..11

2.4. Principle of reflectron ToF instruments (reproduced with permission from ref. 76). The filled circle represents the faster moving ion ……………………………………….13

2.5. Principle of continuous and delayed extraction……………………………………..14

2.6. Microchannel plate (MCP) detector…………………………………………………16

3.1. Bruker utraFlex III mass spectrometer (reproduced with permission from ref. 84). IS1 and IS2 are ion source lenses; PCIS is the precursor ion selector (also called timed ion + + selector, TIS); P1 and P2 are two ion families, each composed of a precursor ion and its fragments (a precursor ion and its fragments move with the same velocities, as a “family”, through the field-free drift region of the short linear ToF tube; after mass- selection (by PCIS) and LIFT post-acceleration, the ions within a family are disphersed in the reflectron ToF by their m/z values. PLMS is the “post LIFT metestable suppressor”. It removes any fragments formed after the LIFT event…………………………………....23

4.1. MALDI mass spectrum of α-4-pentenyl-ω-(p-vinylbenzyl) polystyrene; all ions are [M + Ag]+ adducts. The expanded trace shows the peaks for the 14-mer and 15-mer and the corresponding measured monoisotopic m/z ratios; the calculated monoisotopic m/z values are 1749.92 and 1853.98, respectively.58,78……………………………...…….....26

4.2. MALDI-MS2 spectrum of the silverated 19-mer of α-4-pentenyl-ω-(p-vinylbenzyl) polystyrene (m/z 2270.5); the fragment nomenclature is explained in Figure 4.3. The expanded trace shows the types of fragments observed in the medium and upper mass range of the spectrum…………………………………………………………………….27

4.3. Nomenclature scheme for the MS2 fragments from linear polystyrenes and major terminal fragment ions; Ph symbolizes the phenyl substituent. The charge is provided by

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+ Ag (omitted for brevity). Series an, ana, and bn contain the α end group (Rα) and series yn, yna, and zn the ω end group (Rω). The subscript indicates the number of complete or partial repeat units remaining in the fragment ion.27,44………………………………...... 30

4.4. MALDI mass spectra of the macrocyclic polystyrenes prepared by (a) metathesis ring-closure of α-4-pentenyl-ω-(p-vinylbenzyl) polystyrene (cf. Figure 4.1) and (b) after hydrogenation of the double bond of the latter macrocycle; all ions are [M + Ag]+ adducts. The expanded traces show the peaks for the 14-mers and 15-mers and include the corresponding measured monoisotopic m/z ratios; the calculated monoisotopic m/z values are (a) 1721.89 and 1825.95 and (b) 1723.91 and 1827.97, respectively.58,78...….32

4.5. MALDI-MS2 spectra of (a) the silverated 23-mer from the macrocyclic PS obtained by metathesis ring-closure of α-4-pentenyl-ω-(p-vinylbenzyl) polystyrene (m/z 2658.5) and (b) the silverated 16-mer from the hydrogenated macrocycle (m/z 1932.0) and an expanded trace of the m/z 870-900 region of this spectrum……………………………..34

4.6. MALDI mass spectrum of the macrocyclic polystyrene prepared by click cyclization of a telechelic α-propargyl-ω-azido polystyrene that was synthesized by atom transfer radical polymerization (cf. Scheme 4.4); all ions are [M + Ag]+ adducts. The expanded trace shows the peaks for the 11-mer and 12-mer and the corresponding measured monoisotopic m/z ratios; the calculated monoisotopic m/z values are 1418.66 and 1522.73, respectively.4……………………………………………………………………...40

4.7. MALDI-MS2 spectrum of the silverated 11-mer (m/z 1418.6) from the macrocyclic PS obtained by click chemistry of α-propargylisobutyryl-ω-azido polystyrene (cf. Scheme 4.4). The relative intensity axis is expanded 15 times below m/z 1260………………....41

4.8. MALDI-MS2 spectrum of the silverated 17-mer (m/z 1025.7) from a macrocyclic PB obtained by ring-opening metathesis polymerization of 1,5,9-cyclododecatriene.79….....44

4.9. MALDI-MS2 spectrum of the silverated 26-mer from a linear polybutadiene (PB) prepared by living anionic polymerization in cyclohexane, using sec-C4H9Li for initiation 88 and CH3OH for termination; under these, most favorable conditions for 1,4-addition, 1 ~10% of the monomer is added in 1,2-fashion. Series an, dn, and wn designate terminal • fragment ions, and Jn , Kn, and Ln are internal fragment ions resulting from backbiting rearrangements.88 See Figure 4.10 for the definition of the nomenclature and the structures of the major fragment ion series………………………………………………46

4.10. (a) Nomenclature scheme for the MS2 fragment ions from linear polybutadienes and 2 44 (b) major terminal ions in the MS spectrum of silverated C4H9–(CH2CH=CHCH2)26–H + (the Ag ion is omitted for brevity). Series dn and wn arise from C–C bond cleavages within repeat units incorporated in 1,4-fashion. Series an result from C–C bond cleavages at units incorporated in 1,2-fashion.88…………………………...……………………….47

5.1. (a) MALDI mass spectrum of poly(m-dimethylsilylstyrene-co-styrene); all ions are Na+ adducts. (b,c) Expanded views of the (a) low and (b) high mass distribution,

xii containing monomeric products A-C and dimeric (2-armed) products F-H, respectively; in the dimeric product, the m-DMSS comonomers may be located on either of the two arms and arm linking is possible at any of the m-DMSS units. Measured and calculated monoisotopic m/z values are given for the oligomers observed in the expanded views…50

5.2. (a) MALDI mass spectrum of poly(p-dimethylsilylstyrene-co-styrene); all ions are Na+ adducts. (b,c) Expanded views of the (a) low and (b) high mass distribution, containing monomeric products A-E and dimeric (2-armed) products F-L, respectively; in the dimeric product, the p-DMSS comonomers may be located on either of the two arms and arm linking is possible at any of the p-DMSS units. See Table 5.1 for the calculated m/z values of the oligomers contained in the expanded views...... 51

5.3. (a) Fragment ion notation for MS2 products from polystyrenes; (b) examples of the radical ion fragments (left) and internal fragments (right) dominating the low-mass region of MS2 spectra; (c) homologous fragment series containing the initiating (α) or terminating (ω) end group and a methylene group at the other chain end. The subscripts give the overall number of complete or partial repeat units and the superscripts the number of DMSS comonomer units …………………………………………………….55

5.4. Mid-mass region of the MALDI-MS2 mass spectra of [M+Li]+ ions from (a) the homopolymeric polystyrene oligomer C4H9–(styrene)17–H (m/z 1834.2) and (b) the copolymeric (end-capped) oligomer with the block connectivity C4H9–(styrene)11-b-(p- DMSS)2–H (m/z 1534.0). See Figure 5.3c for the structures of an and yn. The b3" fragment (m/z 377) has the connectivity C4H9–(styrene)3–H (the double prime denotes a • saturated chain end, i.e. one more H atom than b3 ).…...... 57

5.5. (a) MALDI mass spectrum of poly(p-dimethylsilylstyrene-b-styrene), viz. polystyrene end-capped with p-DMSS; all ions are Na+ adducts. (b) Expanded view of the low mass distribution, containing monomeric products with mainly 1-6 p-DMSS repeat units; measured and calculated monoisotopic m/z values are given for the oligomers observed in the displayed m/z range. The dimeric (2-armed) products observed in the high mass distribution contain two H atoms less than the dimeric products from poly(p-dimethylsilylstyrene-co-styrene), which was prepared from a mixture of styrene and p-DMSS (cf. Figure 5.2). A likely structure, generated through linking reactions at two Si–H bonds, is given in Figure 5.5a; such double linking is facilitated by the contiguous arrangement of the p-DMSS units in the end-capped copolymer…………...59

5.6. Low-mass region of MALDI-MS2 the mass spectra of [M+Li]+ ions from (a) the homopolymeric polystyrene oligomer C4H9–(styrene)17–H (m/z 1834.2) and (b) the copolymeric (end-capped) oligomer with the block connectivity C4H9–(styrene)11-b-(p- DMSS)2–H (m/z 1534.0). See Figure 5.3b for the structures of radicals and closed-shell species…...... 61 5.7. Mid-mass region of the MALDI-MS2 mass spectra of [M+Li]+ ions from copolymeric 16-mers of poly(p-DMSS-co-styrene) with the comonomer composition (a)

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(styrene)15(p-DMSS)1 (m/z 1788.2) and (b) (styrene)14(p-DMSS)2 (m/z 1846.2). See Figure 5.3c for the structures of an and yn. The b3" fragment (m/z 377) has the connectivity C4H9–(styrene)3–H (the double prime denotes a saturated chain end, i.e. one • 1 more H atom than b3 ). The sign @ points to trace amounts of yn (n = 8-10); the sign # points to trace amount of an (n = 10-11)………………………………………………....63 5.8. Low-mass region of the MALDI-MS2 mass spectra of [M+Li]+ ions from copolymeric 16-mers of poly(p-DMSS-co-styrene) with the comonomer composition (a) (styrene)15(p-DMSS)1 (m/z 1788.2) and (b) (styrene)14(p-DMSS)2 (m/z 1846.2). See Figure 5.3b for the structures of radicals and closed-shell ions………………………….65 5.9. Mid-mass range of the MALDI-MS2 mass spectrum of [M+Li]+ from the copolymeric 14-mer of poly(m-DMSS-co-styrene) with the comonomer composition (styrene)13(m-DMSS)1 (m/z 1580.0). See Figure 5.3c for the structures of an and yn……66 5.10. Low-mass range of the MALDI-MS2 mass spectrum of [M+Li]+ from the copolymeric 14-mer of poly(m-DMSS-co-styrene) with the comonomer composition (styrene)13(m-DMSS)1 (m/z 1580.0). See Figure 5.3b for the structures of radicals and closed-shell species…………………………………...... 67 6.1. MALDI-ToF mass spectrum of linear in-chain substituted PS. A zoom view of the region between the 31-mer and 32-mer is given in the upper right corner. All ions are [M + Ag]+ adducts …………………………………………………………………………..71 6.2. MALDI-MS2 spectrum of the silverated 31-mer (m/z 3529.2) from the linear in-chain substituted PS shown as inset and in Scheme 6.1. (b) Expanded view of the m/z 1500- 2000 region. See scheme 6.2 for the structures of an, anb, bn, and bni……………………73 6.3. MALDI-ToF mass spectrum of the macrocyclic PS with two linker units prepared as shown in Scheme 6.1. A zoomed view of the m/z region containing the 31-mer and 32- mer is given in the upper right corner. All ions are [M + Ag]+ adducts……...... 75 6.4. MALDI-MS2 spectrum of the silverated 31-mer (m/z 3501.2) from the macrocyle with two linker units shown as inset and in Scheme 6.1. The subscripts e and i denote inclusion of the Si linker at the chain end (e) or in an internal location (i), respectively (cf. Scheme 6.3)………………………………………………………………………………77 6.5. MALDI-ToF mass spectrum of the 4-arm star-branched PS produced from a tetrachlorosilane according to reaction sequence in Scheme 6.4. . Zoom in low-mass range upper in the left is between 9-mer and 10-mer and in high-mass region upper in the right is between 32-mer and 33-mer, respectively. Zoomed views of select m/z regions in the low-mass and high-mass distributions are given in the upper left or upper right corners, respectively. All ions ar [M + Ag]+ adducts……………………...…………….81 6.6. MALDI-MS2 spectrum of the silverated 32-mer from the 4-arm star-branched PS shown as inset and in Scheme 6.4 (m/z 3779.49). The total masses of end and central groups are marked on top of the 3 fragment distributions observed……………………………………………………………………………….....82

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6.7. Zoomed MALDI-MS2 spectra of the silverated 32-mer from the star-branched PS shown in Figure 6.6, showing the m/z region of the (a) linaer and (b) 2-armed PS fragments. The main series in these regions are marked by # and *, respectively, and correspond to the linear PS and 2-arm PS fragments shown in Scheme 6.5………....….83 6.8. MALDI-MS2 spectrum of the silverated 33-mer from the 4-arm star-branched PS shown as inset and in Scheme 6.4 (m/z 3883.58)...... 84

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LIST OF SCHEMES

Scheme Page 3.1. Metathesis ring-closure……………………………………………………………...18 3.2. Mechanism of the Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) “Click” chemistry..………………………………………………………………………………..19 4.1. Synthesis of divinyl telechelic polystyrene by living anionic polymerization using pentenyl lithium as initiator and p-vinylbenzyl chloride as terminating electrophile, followed by cyclization of the telechelic chain via metathesis ring-closure with Grubb’s first generation catalyst, viz. bis(tricyclohexylphosphine)benzylidene ruthenium(IV) chloride.58,78 The double bond in the macrocycle generated this way was hydrogenated using Wilkinson’s catalyst, viz. (triphenylphosphine)rhodium(I) chloride.78………………………………………………………………………………...25 4.2. Backbiting in the benzylic radical ions emerging after random homolytic C–C bond cleavages in Ag+-cationized polystyrene chains; the Ag+ ion is not shown for brevity.27,44 Ph abbreviates the phenyl (C6H5) substituent. Rα and Rω designate the α and ω end   groups, respectively; bn are the benzylic radical ions containing Rα and zn the benzylic radical ions containing Rω. Backbiting gives rise to terminal an and yn fragments (from    bn and zn , respectively), as well as to the internal fragments J2 (m/z 302) and K3 (m/z   419). The same internal ions are produced from all bn and zn radical ions that carry enough repeat units to undergo backbiting. The metal ion is bound strongly between the π electrons of two adjacent phenyl groups, leading to charge-remote fragmentation upon MS2 activation.27…………………………………………………………………………28 4.3. Major dissociation pathways of silverated polystyrene macrocycles, commencing with random homolytic C–C bond cleavages within the PS chain. The 1,2-phenyl shift may occur after ring opening or after one or more monomer units have been eliminated. All species are ionized by Ag+ (omitted for brevity)…………………………………….36 4.4. Synthesis of α-propargyl-ω-bromo polystyrene by atom transfer radical polymerization (ATPR) using trimethylsilyl (TMS) protected propargyl 2- bromoisobutyrate as initiator and Cu(I)Br plus N,N,N′,N′′,N′′- pentamethyldiethylenetriamine (PMDETA) as catalyst. The bromine-terminated product was reacted with NaN3 in dimethyl formamide (DMF) to obtain propargyl-azide telechelic polymer, the TMS group was removed with tetrabutylammonium fluoride (TBAF), and the resulting product was cyclized via “click” chemistry with Cu(I)Br/PMDETA as catalyst.4,56…………………………………………..……………39

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4.5. Major dissociation pathways of silverated polystyrene macrocycles, commencing with selective homolytic bond cleavages within the linker substituent. All species are ionized by Ag+ (omitted for brevity)…………………………………………………….42

4.6. Dissociation of silverated polybutadiene macrocycles via homolytic CH2–CH2 cleavages yielding two allylic radicals at the chain ends. All species are ionized by Ag+……………………………………………………………………………………….45 6.1. Reaction scheme for the linear in-chain substituted PS and the PS macrocycle with two linker units analyzed………………………………………………………………...70 6.2. Major fragmentation pathways of linear in-chain substituted PS…………………...74 6.3. Major dissociation pathways of the silverated PS macrocycles with two linkers, one inert (C8H14) and one with weak bonds in α and β positions to a Si atom. Random homolytic cleavages within the PS chains create diradicals that can ultimately yield the same types of fragments as those generated after initial Cα-Cβ bond cleavage………….78 6.4. Reaction sequence to the 4-arm star-branched PS analyzed………………………...80 6.5. Major fragmentation pathways for a star-branched PS with 4 arms attached to Si atom. These reactions involve arm eliminations, commencing with Si-Cα bond cleavage…………………………………………………………………………………..85 6.6. Charge-remote free radical chemistry accounting for all major MS2 fragments formed from silverated 4-arm star-branched polystyrene oligomers. The second PS arm may be cleaved from the same Si site as the first (shown here) or from the Si site still containing 2 PS arms (as shown in Scheme 6.5). The former pathway leads to a – Si(CH3)H2 terminal group which reconciles more readily the minor bn + 30 and 2-arm bn - 44 fragments. The minor 2-arm fragment possessing an extra 90-Da moiety (see text) nd α β arises by cleavage of the 2 arm at the C -C bond, giving rise to a –Si(CH3)(H)CH2Ph terminal group which is 90 Da heavier than –Si(CH3)H2...... 86

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ABBREVIATIONS

ATRP Atom-transfer radical polymerization CI Chemical ionization DE Delayed extraction EI Electron impact ESI Electrospray ionization FAB Fast atom bombardment FD Field desorption FI Field ionization FTICR Fourier transform ion cyclotron resonance GPC Gel permeation chromatography MS Mass spectrometry m/z Mass-to-charge ratio MALDI-MS2 Matrix-assisted laser desorption ionization tandem mass spectrometry MALDI Matrix-assisted laser desorption ionization m-DMSS Meta-dimethylsilyl styrene MCP Microchannel plate MWD Molecualr weight distribution NMR Nuclear magnetic resonance

Mn Number-average molecular weight 1D-MS One-dimensional mass spectrometry p-DMSS Para-dimethylsilyl styrene ppm Part per million

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PB Polybutadiene PDI Polydisphersity index PS Polystyrene PCIS Precursor ion selector PIE Pulsed ion extraction QIT Quadrupole ion trap Q Quadrupole SIMS Secondary ion mass spectrometry SEC Size exclusion chromatography MS2 Tandem mass spectrometry ToF Time-of-flight ToF/ToF Time-of-flight/time-of-flight DCTB Trans-2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2- enylidene]malonitrile 2D-MS Two-dimensional mass spectrometry

Mw Weight-average molecular weight

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CHAPTER I

INTRODUCTION

Most of the materials we use today are made from synthetic polymers or largely contain synthetic polymers. The manufacture of essentially all common household or personal items such as tires, televisions, computers, cameras, glasses, personal care products, clothing etc involves synthetic polymers. This multitude of applications has promoted the development of many different synthetic techniques for the preparation of synthetic polymers.1 Among these, living and controlled polymerization methods offer superior control of the macromolecular properties of the resulting products, in particular their composition, molecular weight, molecular weight distribution, and architecture.1

Moreover, living and controlled polymerizations usually produce polymers of low polydisphersity, which are ideally suitable for mass spectrometry studies, because their narrow molecular weight distribution minimizes mass discrimination effects during ionization, transmission, and detection.2 The polymers to be discussed in this dissertation were prepared by living anionic polymerization3 and atom-transfer radical polymerization

(ATRP).4 Mass spectrometry can provide analytical information not available by other methods. For example, nuclear magnetic resonance (NMR) spectroscopy unveils precise information about polymer structure but may not detect impurities because high sample concentrations are needed compared to mass spectrometry where nanomole to picomole

1 quantities are enough to obtain observable signals. Gel permeation chromatography

(GPC), another technique used widely to characterize synthetic polymers, only provides average molecular weight distribution (MWD) information such as the number-average molecular weight (Mn) and weight-average molecular weight (Mw), from which the polydisphersity index (PDI = Mw /Mn) can be derived; whereas mass spectrometry can separate single oligomers, which is essential for elucidating polymer structures and identifying any impurities present in the analyzed sample.

Mass spectrometry (MS) studies begin with conversion of the sample to gas-phase molecular ions. Among the first ionization methods used for synthetic polymers were field desorption (FD)/field ionization (FI), fast atom bombardment (FAB) and secondary ion mass spectrometry (SIMS). These methods were successfully applied to small oligomers, but their limitation is that they are “hard”, meaning that they produce both intact analyte ions and fragments, thus, making spectral interpretation difficult.2

The invention of soft ionization techniques, especially matrix-assisted laser desorption/ionization (MALDI)5,6 and electrospray ionization (ESI),7 has significantly broadened the applicability of mass spectrometry to synthetic polymers.8-16 These soft ionization methods cause no or little fragmentation to large molecules, thereby permitting the observation of intact synthetic polymers as [M + X]+ or [M – X]- ions, where X represents a cation added or removed to form a quasimolecular cation or anion, respectively. The mass spectrometer measures the masses of these ions, from which molecular compositions can be deduced. Additional structural information can be obtained by tandem mass spectrometry (MS2) experiments (2D mass spectrometry), in which specific oligomers (n-mers) are selected and induced to decompose to structurally

2 diagnostic fragments. MS2 studies have been reported on a number of homopolymers and copolymers.17-51

Synthetic polymers with unique structures, that endow them with specific chemical and physical properties, are desired for the manufacture of industrial, consumer, and biomedical products. The development of such materials is therefore a major task in polymer science.52 Cyclic polymers have gained attention in the polymer community because their lack of end groups leads to reduced hydrodynamic volumes, resulting in distinctive adhesion, viscoelastic, and dispersion properties.53 Polymers with macrocyclic architectures can be efficiently prepared by cyclization of properly substituted α,ω- difunctional chains.54-58 Unequivocal characterization of the macrocyclic architectures is challenging, however, because isomeric linear structures, terminated by a double bond, are possible. Linear and cyclic polymeric isomers have been distinguished by ion mobility mass spectrometry.59 In chapter IV, the applicability of tandem (2D) mass spectrometry (MS2) for differentiating cyclic from linear polymers will be discussed.

Copolymers are composed of two or more different comonomers. Depending on the type of monomers and synthetic approach used, sequences with block, tapered, alternate, or random architecture may be formed.1 The analysis of such materials by 1D mass spectrometry (MS) can be problematic, as the molecular weight and end group distributions are convoluted by comonomer content distributions.60-61 With the soft ionization techniques matrix-assisted laser desorption ionization (MALDI)5,6 and electrospray ionization (ESI),7 and a reasonable mass accuracy (≤ 15 ppm), single-stage

(1D) MS can provide information about end groups, byproducts, and comonomer composition,60-61 but insight on the sequence(s) produced is difficult to extract from such

3 spectra, requiring elaborate modeling of the observed ion intensities by statistical methods which is feasible for low molecular weight chains only.62-65 More definitive sequence information can be obtained by MS2 analysis of specific oligomers. MS2 has been applied to differentiate block from random copolymers66-70 and to confirm the presence of random comonomer arrangements in short polyester and poly(ester amide) oligomers.71-73 In this dissertation, MALDI has been combined with MS and MS2 to examine the copolymer distributions, architectures, and sequences generated by living anionic polymerization of styrenic monomers (chapter V).

The styrenic comonomer reactants were mixtures of mainly styrene plus either meta-dimethylsilylstyrene (m-DMSS) or para-dimethylsilylstyrene (p-DMSS), which

74 were copolymerized to create oligomers with dimethylsilyl pendants, –Si(CH3)2H; these polymeric silyl hydrides can serve as macromonomers to synthesize star-branched or comb-type polymers via hydrosilation.75 Due to the high reactivity of Si–H bonds, the macromonomer synthesis is accompanied by side reactions. As will be shown in chapter

V, MS methods are ideally suitable for the detection and identification of the byproducts as well as for the characterization of the comonomer composition of the main (desired) products. Further, the utility of MS2 for not only distinguishing copolymer sequences but also determining specific comonomer positions along a chain will be illustrated for the first time.

Lastly, chapter VI will describe the application of tandem mass spectrometry to characterize centrally functionalized linear polymer (in-chain functionalization), macrocycle with two linker groups, and four-arm star-branched polymers.

4

CHAPTER II

MASS SPECTROMETRY BACKGROUND

2.1. Ionization techniques

For mass spectrometry analysis of any sample to be possible, ions must be first created, either positive or negative. Common ionization methods are: electron impact

(EI), chemical ionization (CI), field desorption or ionization (FD or FI), secondary ion mass spectrometry (SIMS), matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI). Only MALDI will be discussed in this dissertation because it was the only ionization technique utilized.

2.2. MALDI

The initial prep of the MALDI process involves mixing analyte with excess matrix, normally in the molar ratio of 1:1000-10000 (analyte : matrix). The matrix is a small organic compound with a strong absorption at the laser wavelength being used.

Most matrices are aromatic molecules with OH or COOH groups.76-77 The MALDI experiment involves two processes. The first step is desorption; the matrix plays an important role by being desorbed from the surface and pulling along the sample with it in the gas phase. The second step is ionization; again the matrix is involved in this process

5 by assisting the transfer of a proton or other cationization agent (e.g. Na+, Ag+, and etc.) to the analyte (see Figure 2.1).

Figure 2.1. MALDI principle (reproduced with permission from ref. 76).

In MALDI analysis, matrix selection (see Table 2.1) is very important. The matrix must accomplish the following functions: first, it must protect the sample from being damaged by the laser power. Second, the matrix must separate the analyte molecules as far apart as possible from each other to prevent formation of analyte clusters. This is achieved by mixing the analyte with an excess molar amount of matrix.

6

Table 2.1. Common MALDI matrices (reproduced with permission from ref. 76).

In order to obtained intense MALDI signals, different sample preparation techniques have been developed. The most common and widely used sample preparation technique is the dried-droplet method. In this method, the matrix, sample, and cationization agent (if it is needed) are mixed together and a small aliquot of this mixture is applied on the steel MALDI plate and allowed to dry at room temperature before it is inserted in the instrument. Another technique is the sandwich method. For this method, a mixture of matrix and cationizing agent is applied on the bottom, then the sample, and on top a second layer of matrix and salt mixture is applied again.

The most common lasers in MALDI instruments are UV-lasers. Such lasers are relatively cheap to replace and maintain as compared to IR-lasers. Table 2.2 below shows a list of both UV- and IR-lasers that have been used in MALDI mass spectrometers.

7

Table 2.2. Common lasers used for MALDI experiment (reproduced with permission from ref. 76).

MALDI is widely used for polymers and biopolymers because of its softness.

Normally, intact molecules are observed with little or no fragmentation. Moreover,

MALDI generally produces singly charged species and this makes spectral interpretation more straight forward as compared to ESI which can produces multiple charge species thus leading to very complex spectra for synthetic polymers due to overlapping charge and molecular weight distributions.

2.3. Mass analyzers

After the creation of ions, the next step is their mass analysis by separation based on their mass-to-charge (m/z) ratio. This is accomplish by the mass analyzer. There are different types of mass analyzers: quadrupole (Q), quadrupole ion trap (QIT), time of flight (ToF), magnetic, fourier transform ion cyclotron resonance (FTICR), and orbitrap.

In this dissertation, only the ToF will be discussed which was the only mass analyzer utilized.

8

2.3.1. Mass resolution

Mass resolution is very important in mass analysis because it is directly related to the mass accuracy of the mass analyzer. The basic resolution requirement for simple mass analysis is the ability distinguish ions differing by one mass unit (m/z) and most mass analyzers, including ToF mass analyzer, provide such unit mass resolution. Figure 2.2 below illustrates the concept of resolving power, showing peaks m1 and m2 resolved at

10% and 80% valley.

9

Figure 2.2. Resolving power (reproduced with permission from ref. 76).

2.3.2. Time of fight (ToF) mass analyzer

The operation of ToF mass analyzers is based on the physics of ion motion. After the ions have been formed in the MALDI source, they are accelerated and travel at constant velocity v inside a field-free tube that has fixed lenght L until they arrive at the detector. The arrival time at the detector t is measured and converted to m/z, see Figure

2.3 below.

10

Figure 2.3. Linear time of fight principle (reproduced with permission from ref. 76).

An ion with mass m and total charge q = ze that is accelerated by a potential Vs

(20-25 kV) out of the source, gains the potential energy Eel which becomes its kinetic

76 energy Kk (see Equation 1).

2 Kk = mv /2 = qVs = zeVs (1)

The accelerated ion “flies” through a field-free region of length L to reach the detector. The fight time t depends on the ion velocity v (see Equation 2)76 which is also related to mass-to-charge ratio (m/z) (see Equation 1).76 t = L/v (2)

Combining Equation 1 and Equation 2 results in Equation 3, which relates the time-of- flight to the m/z ratio.

11

2 2 1/2 t = m/z (L /2eVs) (3)

In theory, ToF instruments have no upper mass limit as long as the analyte ions can fly and reach the detector. Linear ToF instruments suffer from poor resolution because ions of the same m/z may leave the source at different times or with different initial kinetic energies, which result in slightly different final kinetic energies and different arrival times at the detector. To correct this problem, a reflectron and delayed extraction (DE)

(or pulsed ion extraction (PIE) or time lag focusing) must be used.

2.3.2.1. Reflectron

The reflectron corrects the flight times of ions of the same m/z leaving the source with slightly different initial kinetic energies. The initially faster ion penetrates deeper and thus spends more time inside the reflectron before it is re-accelerated out of the reflectron back into the field-free region of the flight tube. Conversely, the slower ion penetrates less in the reflectron before it is re-accelerated into the field-free flight tube, which enables it to catch up with the fast moving ion so that they reach the reflector detector at the same time. The result is a resolution greater than 20,000 and a mass accuracy of less than 10 ppm (see Figure 2.4).

12

Figure 2.4. Principle of reflectron ToF instruments (reproduced with permission from ref. 76). The filled circle represents the faster moving ion.

2.3.2.2. Delayed extraction (DE) or pulsed ion extraction (PIE) or time lag focusing

The principle of delayed extraction is illustrated in Figure 2.5. The top portion of the Figure shows two ions with the same m/z value leaving the source with slightly different initial kinetic energy and subjected to continuous extraction, which causes them to arrive at the detector at different times; this results in peak broadening and poor resolution. The bottom part of Figure 2.5 shows the same two ions with different initial kinetic energy; now, these ions are first allowed to drift in a field-free region inside source before applying an extraction pulse after a small delayed time (~10-7-10-6 sec).

The ion closer to the source gains more energy and travels faster after acceleration, whereas the ion that travelled further gains less energy and moves more slowly. The initially slower ion moves faster and the initially faster ion travels more slowly in field-

13 free region of the ToF mass analyzer, so that they reach the detector at the same time which improves resolution as well as mass accuracy.

Figure 2.5. Principle of continuous and delayed extraction (reproduced with permission from ref. 76).

14

2.4. Detectors

After the ions are separated by their m/z in the mass analyzer, they are detected based on electrical signals they produce by striking a detection system. There are several types of widely used detectors: electron multipliers, array detectors (e.g. microchannel plate detectors), and photon multipliers.76 Only the microchannel plate detector was utilized in this dissertation and will therefore be briefly described.

2.4.1. Microchannel plate (MCP) detector

MCP detectors are composed of many small channels with about 5-25 μm in diameter and center-to-center distances between adjacent channels of 6-32 μm (see

Figure 2.6). The surfaces of the channels are coated with a semiconductor material, which generates secondary electrons when struck by an ion beam. The cascade effect inside a channel (see Figure 2.6) causes the ejection of a large number of electrons per striking ion (about 105). To increase this cascade effect more plates can be connected in a parallel arrangement, yielding gains of about 108. The MCP detector is suitable for the detection of ions generated by a pulsed technique, such as MALDI, that creates ions with a spatial distribution; as long as all ions with the same m/z ratio reach the detector at the same time, it does not matter where they hit on the detector. They can be detected simultaneously.

15

Figure 2.6. Microchannel plate (MCP) detector (reproduced with permission from ref. 76).

16

CHAPTER III

MATERIALS AND EXPERIMENTAL PROCEDURES

3.1. Materials

3.1.1. Linear and cyclic polymers

The linear and cyclic polymers were synthesized in the laboratory of Prof. R. P.

Quirk (by S-F. Wang and V. S. Chavan) in the Department of Polymer Science at The

University of Akron and the laboratory of Prof. S. M. Grayson (by B. A. Laurent) in the

Department of Chemistry at Tulane University. The synthetic routes to the samples analyzed have been described in detail elsewhere.4,58,78-79 Briefly, α,ω-difunctional polystyrenes57,58,78 were prepared by living anionic polymerization58,78 or atom transfer radical polymerization57 and cyclized by metathesis ring-closure58,78 (see Scheme 3.1) or click chemistry57,80 (see Scheme 3.2). Linear and cyclic polybutadienes were prepared by conventional living anionic polymerization of butadiene79 and ring-opening metathesis polymerization of 1,5,9-cyclododecatriene,79,81 respectively. The mass spectrometry studies on these polymers will be discussed in chapter IV of this dissertation.

17

Grubb’s cat.

Linear precursor

Macrocycle

Scheme 3.1. Metathesis ring-closure

18

Scheme 3.2. Mechanism of the Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) “Click” chemistry (reproduced with permission from ref. 80).

19

3.1.2. Polystyrene copolymers

The styrene-co-p-DMSS or m-DMSS copolymers were synthesized in the laboratory of Prof. R. P. Quirk (by J. E. Janoski) in the Department of Polymer Science at

The University of Akron. Polystyrene homopolymer and copolymers of styrene (Aldrich) and either m- or p-DMSS were prepared by living anionic polymerization in cyclohexane

(Aldrich) using sec-BuLi (FMC, Charlotte, NC) for initiation and MeOH (Aldrich) for termination. The initiator was added to a solution of the monomer(s) and the polymerization was allowed to proceed for 1-3 hours before quenching with MeOH; the precipitated polymers were dried in vacuo.74 In addition to poly(p-DMSS-co-styrene) copolymers, polystyrene end-capped with p-DMSS was also synthesized by reacting homopolymeric polystyryl lithium with four equivalents of p-DMSS before termination with methanol. All polymerizations were carried out in sealed, all-glass apparatus using standard high-vacuum techniques.82,83 The detailed synthetic procedure of the polymers discussed in this study and of the m- and p-DMSS comonomers has been described elsewhere.74 The characterization of these polymers by mass spectrometry will be discussed in chapter V.

3.1.3. Linear in-chain functionalized precursor, cyclic with two linker units, and four star polystyrenes

The linear in-chain, cyclic with two linker units, and four-arm star-branched polymers were synthesized in the laboratory of Prof. R. P. Quirk (by S-F. Wang and Q.

He) in Department of Polymer Science at The University of Akron. The polymers were prepared by living anionic polymerization and the cyclization was achieved with the 1st

20 generation Grubb’s catalyst. The mass spectrometry characterization of such polymers will be presented in chapter VI of this dissertation.

3.2. MALDI experimental procedures

MS and MS2 experiments were performed on a Bruker Ultraflex III MALDI tandem time-of-flight (ToF/ToF) mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a Nd:YAG laser emitting at 355 nm.48 DCTB, viz. {2-[(2E)-3-(4-tert- butylphenyl)-2-methylprop-2-enylidene]malonitrile} (99%+; Santa Cruz Biotechnology,

Santa Cruz, CA ), and silver trifluoroacetate (98%; Aldrich, Milwaukee, WI) served as matrix and cationization salt (for PS), respectively. For styrene/p-DMSS or m-DMSS copolymers, sodium trifluoroacetate (99%+; Aldrich, Milwaukee, WI) or lithium trifluoroacetate (95%; Aldrich) served as cationization salts. Na+ was used in MS mode and Li+ in MS2 mode; Li+ adduction increases the complexity of single-stage mass spectra due to co-ionization with adventitious Na+, but lithiated ions give more abundant fragment ions (better signal/noise ratio) in tandem mass spectra. Silver salts, which are normally employed as cationizing agents for polystyrenes, cannot be used because Ag+ oxidizes the Si–H group to Si–OH.84 Solutions of the matrix (20 mg/mL), cationizing salt

(10 mg/mL), and polymer sample (10 mg/mL) were prepared in THF (99.9%; Aldrich).

These solutions were mixed in the ratio of matrix: polymer: cationizing agent (10:2:1), and 0.5-1.0 µL of the final mixture were applied to the MALDI sample target and allowed to dry at ambient conditions before spectral acquisition. This sample preparation protocol led to the formation of [M+Na]+ or [M + Li]+ or [M+Ag]+ ions after MALDI.

21

3.3. MALDI instrumentation

A mass spectrometer usually has five basics components: sample introduction system, ion source, mass analyzer, detector, and data processing system. The sample introduction system in MALDI analysis is usually a target plate, on which sample is spotted and allowed to dry before the target is inserted in the ion source. The next component is the MALDI source; this is the region where ions are created, either negative or positive. The ions formed are accelerated into the ToF tube, where they are separated based on m/z values. MCP detection follows and the data are displayed in the form of mass spectrum (relative intensity vs. m/z) on the computer monitor. The instrument utilized for the work in this dissertation was the Bruker MALDI-ToF/ToF

UltraFlex III.

The UltraFlex III ToF/ToF mass spectrometer (see Figure 3.1) is composed of a short linear ToF tube, interfaced axially with a reflectron ToF device.48,85 MS2 spectra were acquired using the LIFT technique, which involves raising the laser intensity to induce fragmentation in the linear ToF part, isolation of the desired precursor ion and its fragments (“ion family”) by a high-resolution timed ion selector (TIS), and post- acceleration (“lifting”) of the selected ion family for mass analysis in the reflectron ToF segment. The IS1, IS2, and lens potentials in the ion source were set at 8.00, 7.15, and

3.60 kV, respectively, the LIFT 1 and 2 potentials at 19.00 and 2.90 kV, respectively, and the reflectron 1 and 2 lenses at 29.50 and 13.85 kV. For single-stage (1-D) mass spectra, the TIS and LIFT devices were grounded, and the IS1, IS2, source lens, reflectron 1, and reflectron 2 potentials were set at 25.03, 21.72, 9.65, 26.32, and 13.73 kV, respectively.

22

Figure 3.1. Bruker utraFlex III mass spectrometer (reproduced with permission from ref. 85). IS1 and IS2 are ion source lenses; PCIS is the precursor ion selector (also called + + timed ion selector, TIS); P1 and P2 are two ion families, each composed of a precursor ion and its fragments (a precursor ion and its fragments move with the same velocities, as a “family”, through the field-free drift region of the short linear ToF tube; after mass- selection (by PCIS) and LIFT post-acceleration, the ions within a family are disphersed in the reflectron ToF by their m/z values. PLMS is the “post LIFT metestable suppressor”. It removes any fragments formed after the LIFT event.

23

CHAPTER IV

DIFFERENTIATION OF LINEAR AND CYCLIC POLYMER ARCHITECTURES BY

MALDI TANDEM MASS SPECTROMETRY (MALDI-MS2)

The work in this chapter was published in J. Am. Soc. Mass Spectrom. (2013) and is reproduced here with permission from the publisher.

4.1. Linear Polystyrene

Living anionic polymerization of styrene using 4-pentenyllithium as initiator and p-vinylbenzyl chloride as the terminating electrophile gives rise to a telechelic polystyrene (PS) that carries 4-pentenyl and p-vinylbenzyl substituents at the α and ω chain ends, respectively (see structure in Figure 4.1, 4.2, and Scheme 4.1).58,78 Reaction of 1.74 mmol initiator with 41.5 mmol monomer and 17.4 mmol terminating agent led to a polymer with an average molecular weight (Mn) of 2800±140 Da, as determined by size exclusion chromatography (SEC) with light scattering detection.78 The corresponding

MALDI mass spectrum (Figure 4.1) shows a narrow molecular weight distribution in the desired molecular weight range and essentially one series of [M + Ag]+ ions with the expected composition, in accord with the α-pentenyl-ω-vinylbenzyl chain end functionality needed for subsequent cyclization via metathesis ring-closure (vide infra).

24

(1) n

(2)

C43H72Cl2P2Ru

H2, PPh3

RhCl(PPh3)3

Scheme 4.1. Synthesis of divinyl telechelic polystyrene by living anionic polymerization using pentenyl lithium as initiator and p-vinylbenzyl chloride as terminating electrophile, followed by cyclization of the telechelic chain via metathesis ring-closure with Grubb’s first generation catalyst, viz. bis(tricyclohexylphosphine)benzylidene ruthenium(IV) chloride.58,78 The double bond in the macrocycle generated this way was hydrogenated using Wilkinson’s catalyst, viz. (triphenylphosphine)rhodium(I) chloride.78 (reproduced with permission from ref. 26).

25

1853.89 1749.83

1760 1800 1840 m/z

1000 1500 2000 2500 3000 3500 4000 m/z

Figure 4.1. MALDI mass spectrum of α-4-pentenyl-ω-(p-vinylbenzyl) polystyrene; all ions are [M + Ag]+ adducts. The expanded trace shows the peaks for the 14-mer and 15- mer and the corresponding measured monoisotopic m/z ratios; the calculated monoisotopic m/z values are 1749.92 and 1853.98, respectively.58,78 (reproduced with permission from ref. 26).

Figure 4.2 shows the MALDI-MS2 spectrum of the silverated 19-mer of the divinyl-terminated polystyrene. This spectrum includes the typical fragments observed from the metal ion adducts of linear, chain-end functionalized polystyrenes, viz. abundant

• • • • radical ions carrying either the α or the ω chain end (b1 , b2 , z1 ) and internal ions (J2 , K3) in the low-mass region (see Scheme 4.2 for their structures), as well as two homologous series of relatively sizable fragments (an, yn) across the medium- and high-mass region, each containing one original end group and one newly produced methylene end group

(Figure 4.3).44 This fragmentation pattern arises from random, charge-remote homolytic

C–C bond cleavages along the polymer backbone which create charged radicals that may decompose further by backbiting rearrangements (Scheme 4.2), bond migrations (phenyl shifts), and β scissions.22,27-28

26

a8 a9

• 917

b1 1021

280 y7 • z1 • z2 b 951 8

b7 z7 a9a y8a 314

J • K3 903

2 1007

937 945 979 • b2 419 900 920 940 960 980 1000 1020 m/z • b3 2270.5 384 an (n = 4-20) ^ yn (n = 2-17)

488 * 207 * * ^ ^ ^ *^ *^ * ^ *^ *^ *^ *^ *^ *^ *^ *^ *^ *^ * * 250 500 750 1000 1250 1500 1750 2000 m/z

Figure 4.2. MALDI-MS2 spectrum of the silverated 19-mer of α-4-pentenyl-ω-(p- vinylbenzyl) polystyrene (m/z 2270.5); the fragment nomenclature is explained in Figure 2. The expanded trace shows the types of fragments observed in the medium and upper mass range of the spectrum. (reproduced with permission from ref. 26).

27

• • bn or zn

• • an-2 or yn-2 bn-3 or zn-3 + +

• J2 K 3

Scheme 4.2. Backbiting in the benzylic radical ions emerging after random homolytic C– C bond cleavages in Ag+-cationized polystyrene chains; the Ag+ ion is not shown for 27,44 brevity. Ph abbreviates the phenyl (C6H5) substituent. Rα and Rω designate the α and   ω end groups, respectively; bn are the benzylic radical ions containing Rα and zn the benzylic radical ions containing Rω. Backbiting gives rise to terminal an and yn fragments    (from bn and zn , respectively), as well as to the internal fragments J2 (m/z 302) and K3   (m/z 419). The same internal ions are produced from all bn and zn radical ions that carry enough repeat units to undergo backbiting. The metal ion is bound strongly between the π electrons of two adjacent phenyl groups, leading to charge-remote fragmentation upon MS2 activation.27 (reproduced with permission from ref. 26).

28

Figure 4.3 explains the nomenclature used and summarizes the structures of the fragments generated from linear polystyrenes.44 Initial C–C bond breakup in the PS

• • • • backbone yields truncated chains with benzylic (bn , zn ) and primary (an , yn ) radical sites at the chain ends, which can undergo unzipping, occurring by consecutive monomer losses via β C–C bond scissions, as well as loss of a hydrogen or phenyl radical to

27-28,44 produce six different fragment ion series, viz. an, ana, bn, yn, yna, and zn (Figure 4.3).

• • • Only very small benzylic radical ions survive intact (b1 , b2 , z1 ), indicating extensive (for the benzylic) or complete (for the primary radicals) depolymerization by unzipping. The longer lifetimes of the benzylic radicals also permit the occurrence of hydrogen rearrangements that move the radical site to a more stable internal position (backbiting), cf. Scheme 4.2; subsequent C–C bond cleavages provide an alternative route to the

• 27,44 terminal series an and yn and coproduce the internal fragments J2 and K3. The high

• 2 relative intensities of J2 and K3 in the low-mass range of the MS spectrum and of the an/yn doublets in the medium- and high-mass ranges identify backbiting as the major dissociation pathway of the incipient charged radicals emerging after the initial PS backbone breakup. It is noteworthy that the relative abundances of the yn series are lower than those of the an series, consistent with facile elimination of the ω end group which is attached to the polymer through a bond connecting two benzylic C atoms.28

29

y3 z3 y2 z2 y1 z1

a1 b1 a2 b2 a3 b3

X = Ph an zn X = H ana

bn X = Ph yn X = H yna

Figure 4.3. Nomenclature scheme for the MS2 fragments from linear polystyrenes and major terminal fragment ions; Ph symbolizes the phenyl substituent. The charge is + provided by Ag (omitted for brevity). Series an, ana, and bn contain the α end group (Rα) and series yn, yna, and zn the ω end group (Rω). The subscript indicates the number of complete or partial repeat units remaining in the fragment ion.27,44 (reproduced with permission from ref. 26).

4.2. Cyclic Polystyrenes

The α,ω-divinyl polystyrene was cyclized by metathesis ring-closure using the

Grubb’s catalyst bis(tricyclohexylphosphine)benzylidene ruthenium (IV) chloride;81 the macrocycle resulting from this reaction is depicted in Figure 4.4a. The MALDI mass spectrum (Figure 4.4a) shows only one narrow distribution of [M + Ag]+ ions with the desired composition. The silverated n-mers from the cyclic PS appear 28 m/z units lower than the same n-mers from the linear precursor (cf. Scheme 4.1), in agreement with the

30

Grubbs metathesis mechanism. In a subsequent step, the double bond in the macrocycle was hydrogenated using (triphenylphosphine)rhodium(I) chloride (Wilkinson’s catalyst),78 cf. structure in Figure 4.4b. The MALDI mass spectrum of the hydrogenated polymer (Figure 4.4b) shows a monomodal distribution of [M + Ag]+ ions, which are observed 2 m/z units higher than the same n-mers from the original cyclization product, confirming that the double bond within the macrocycle was quantitatively reduced. SEC analysis of the unsaturated and reduced macrocycle with light scattering detection indicated average molecular weights (Mn) of 2700±140 and 2800±140 Da, respectively.58,78 Cyclization and hydrogenation do not change measurably the molecular weight distribution and average molecular weight, as is also evident from the corresponding MALDI mass spectra (cf. Figures 4.1 and 4.4), and in accord with the chemical transformations effected in these reactions (cf. Scheme 4.1).

31

(a)

1826.03 1721.94

1720 1760 1800 m/z

1000 1500 2000 2500 3000 3500 4000 4500 m/z

(b)

1828.00 1723.94

1740 1780 1820 m/z

1500 2000 2500 3000 3500 4000 m/z

Figure 4.4. MALDI mass spectra of the macrocyclic polystyrenes prepared by (a) metathesis ring-closure of α-4-pentenyl-ω-(p-vinylbenzyl) polystyrene (cf. Figure 4.1) and (b) after hydrogenation of the double bond of the latter macrocycle; all ions are [M + Ag]+ adducts. The expanded traces show the peaks for the 14-mers and 15-mers and include the corresponding measured monoisotopic m/z ratios; the calculated monoisotopic m/z values are (a) 1721.89 and 1825.95 and (b) 1723.91 and 1827.97, respectively.58,78 (reproduced with permission from ref. 26).

The MALDI-MS2 spectrum of the silverated 23-mer from the cyclic polystyrene obtained by metathesis ring-closure is markedly different from the MS2 spectrum of the linear precursor, cf. Figures 4.2 and 4.5a. The linear n-mer preferentially yields fragments that are missing one end group (an/yn pairs); in sharp contrast, the cyclic polystyrene mainly generates fragment ions by losses of n times the monomer, (C8H8)n, or n times the monomer plus an additional CH2 unit, which have been labeled by a blue ◊

32 and a red ‡ sign, respectively. Reducing the double bond in the macrocycle does not change this fragmentation pattern, as indicated by the MALDI-MS2 spectrum of the silverated 16-mer of the hydrogenated product, cf. Figure 4.5b. Essentially all fragments above m/z ~450 in Figure 4.5b appear 2 m/z units higher than the analogous fragments in

Figure 4.5a, providing strong evidence that they include the linker unit and that fragmentation occurs within the PS chain.

33

◊ (a)

◊ (ab)n" 2554 ◊

‡ (bb)n" 2450

• ◊ J2 ◊ K ◊

3 ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ◊ 2346 302 ◊

‡ ‡ 2242

419 ‡

2138

2658.5

1188

1292

1396

1500 2034

1604 ‡

979 1083

1708 1812 193 ‡ ‡

1930 ‡ ‡ ‡ 875 ◊ ◊ ◊ ◊ ◊ ‡ ‡ ◊ 771 ◊ ◊ ◊ ◊ ◊ ◊ 500 1000 1500 2000 m/z

(b) ◊ (bb)6"

‡

1828

1932.0 877 • (ab)6" J2 ◊ ◊

302 ◊

891 1724 ◊ (bb) (ab)6 K3 ‡ ‡ 6 193 ‡ ‡ ‡ ◊

‡ ‡ ◊ 1620

419

1516

877

773

981

669

1412

1086

565 1190 ‡ 1308 ‡ ◊ ◊ ◊ ◊ ‡ ‡ ◊ ◊ ◊ ‡ ‡ 200 600 1000 1400 1800 m/z

Figure 4.5. MALDI-MS2 spectra of (a) the silverated 23-mer from the macrocyclic PS obtained by metathesis ring-closure of α-4-pentenyl-ω-(p-vinylbenzyl) polystyrene (m/z 2658.5) and (b) the silverated 16-mer from the hydrogenated macrocycle (m/z 1932.0) and an expanded trace of the m/z 870-900 region of this spectrum. (reproduced with permission from ref. 26).

34

The energetically preferred dissociation within the PS chain is CH2–CH(Ph) bond cleavage. Such a reaction within the PS segment of Ag+-cationized macrocycles creates linear Ag+-cationized diradicals with one primary and one benzylic radical site at their termini, as shown in Scheme 4.3. Successive monomer losses (unzipping) are facile, especially at the primary radical site, based on the behavior of the radical ions formed from linear polystyrenes.27 The unzipping process proceeds until stopped by intramolecular β-H• transfer from one radical to the other, resulting in fragment ions with one saturated and one olefinic chain end (“disproportionation”). Scheme 4.3 shows H• abstraction from the benzylic carbon, which should have a lower energy requirement than

• the alternative H transfer from a CH2 group. The fragment series formed this way has been termed (ab)n'', because one chain end contains a methylene carbon atom (as in an), the other a benzylic carbon atom (as in bn), and one of them is saturated (indicated by

'').27,44

35

C–C bond cleavage

1,2-phenyl shift

(1) loss of ≥ 1 styrene (C8H8) units (2) intramolecular H• transfer

'' (ab)n

(1) loss of CH2=CHCH2Ph (+ n C8H8) (2) intramolecular H• transfer '' (bb)n

Scheme 4.3. Major dissociation pathways of silverated polystyrene macrocycles, commencing with random homolytic C–C bond cleavages within the PS chain. The 1,2- phenyl shift may occur after ring opening or after one or more monomer units have been eliminated. All species are ionized by Ag+ (omitted for brevity). (reproduced with permission from ref. 26).

36

The second major series is accounted for by a 1,2-phenyl migration (see Scheme

4.3) at the primary radical to form a more stable secondary radical that can lose

28 phenylpropene (118 Da) by β C–C bond scission. Phenyl propene contains one CH2 unit more than the monomer. A series of monomer losses may accompany this rearrangement, ultimately leading to a distribution of ring-opened diradical chains with benzylic radicals at both chains ends. Consecutive intramolecular β-H• transfer between the radical sites is again possible to form fragment ions with one saturated and one olefinic chain end; these have been termed (bb)n'' fragments, as they carry benzylic carbons at both chain ends.

In addition to these major dissociation pathways, backbiting at the benzylic

• radicals takes place, as revealed by the ions observed at m/z 302 (J2 ) and 419 (K3); the extent of this rearrangement is, however, significantly lower than in linear polystyrenes, cf. Figures 4.2 and 4.5. The Ag+-cationized diradicals arising after ring-opening could decompose by sequential β-H• elimination to produce fragments with two unsaturated chain ends, viz. series (ab)n and (bb)n, which should appear 2 m/z units lower than series

2 (ab)n'' and (bb)n", respectively. The expanded MS spectrum in Figure 4.5b indeed confirms the occurrence of this process to a very small degree; the alternative, intramolecular H• rearrangement is more competitive because it involves concomitant bond scission and bond formation, thus lowering the corresponding activation energy.

The foregoing discussion clearly shows that polymer architecture can influence the unimolecular chemistry taking place in MS2 experiments. Linear polystyrenes yield fragment ions that contain only one of the original end groups, whereas cyclic polystyrenes mainly decompose by monomer losses. Based on these characteristics,

37 architectural differentiation by MS2 is possible. To test whether this approach is generally applicable, a PS macrocycle with different linking connectivity was investigated (see structure in Figure 4.6 and Scheme 4.4).

The new polystyrene macrocycle was synthesized according to the four-step procedure summarized in Scheme 4.4.4 First, atom transfer radical polymerization

(ATPR) was utilized to form a linear polymer with propargyl isobutyrate and bromine substituents at the α and ω chain ends, respectively. Then, substitution of the bromine with an azide end group followed. In the final step, intramolecular azide-alkyne cycloaddition in the presence of Cu(I) catalyst (click chemistry) was performed to obtain a PS macrocycle that incorporates a linking unit containing both ester and triazole functionalities. The MALDI mass spectrum of the final product (Figure 4.6) contains one

Gaussian-shaped series of [M + Ag]+ ions with the expected composition, attesting the formation of a product with high purity and narrow molecular weight distribution (Mn =

1800±90 Da according to SEC).4,56

38

Cu(I)Br n + PMDETA

NaN3 DMF

Cu(I)Br TBAF

PMDETA THF

Scheme 4.4. Synthesis of α-propargyl-ω-bromo polystyrene by atom transfer radical polymerization (ATPR) using trimethylsilyl (TMS) protected propargyl 2- bromoisobutyrate as initiator and Cu(I)Br plus N,N,N′,N′′,N′′- pentamethyldiethylenetriamine (PMDETA) as catalyst. The bromine-terminated product was reacted with NaN3 in dimethyl formamide (DMF) to obtain propargyl-azide telechelic polymer, the TMS group was removed with tetrabutylammonium fluoride (TBAF), and the resulting product was cyclized via “click” chemistry with Cu(I)Br/PMDETA as catalyst.4,56 (reproduced with permission from ref. 26).

39

1522.62 1418.56

1420 1460 1500 m/z

1000 1500 2000 2500 3000 m/z

Figure 4.6. MALDI mass spectrum of the macrocyclic polystyrene prepared by click cyclization of a telechelic α-propargyl-ω-azido polystyrene that was synthesized by atom transfer radical polymerization (cf. Scheme 4.4); all ions are [M + Ag]+ adducts. The expanded trace shows the peaks for the 11-mer and 12-mer and the corresponding measured monoisotopic m/z ratios; the calculated monoisotopic m/z values are 1418.66 and 1522.73, respectively.4 (reproduced with permission from ref. 26).

Figure 4.7 depicts the MALDI-MS2 spectrum of the silverated 11-mer from the product of click cyclization. Extensive dissociation by loss of methylene triazole is observed (m/z 1388), consistent with easy homolysis of the allylic C–O and C–N bonds in the linker unit introduced by click chemistry at the internal energies accessed under

MALDI-MS2 conditions (cf. Scheme 4.5). These homolytic cleavages result in a silverated diradical containing a carboxy radical site, as well as a styryl radical site that can depolymerize (unzip); indeed, the fragment at m/z 1388 dissociates further by losses of n times styrene monomer, (C8H8)n, as did the diradicals arising from cyclic polystyrenes with a stable linker moiety (cf. Figures 4.5 and 4.7). Depolymerization proceeds until β-H• abstraction from the terminal benzyl site by the carboxy radical

40 generates closed-shell ions, viz. series (ab)n'' (cf. Scheme 4.5). The polystyrene chains of this series are terminated with one saturated methylene carbon atom (attached to the isobutyryl group) and one unsaturated benzylic carbon atom and, thus, have quite similar structural attributes to those from the macrocycles with a stable linker group (cf. Schemes

4.3 and 4.5).

◊ ◊

◊ (ab)n" (n = 7-11)

1234 1338 * (ab)n (n = 5-11) 81 Da

• ◊

193 J 2 ◊ K *

3 ◊

1130 1363 302 * 1418.6

* 1025

1292

419

921

815 1315 711 * * * 200 600 1000 x15 m/z

Figure 4.7. MALDI-MS2 spectrum of the silverated 11-mer (m/z 1418.6) from the macrocyclic PS obtained by click chemistry of α-propargylisobutyryl-ω-azido polystyrene (cf. Scheme 4.4). The relative intensity axis is expanded 15 times below m/z 1260. (reproduced with permission from ref. 26).

41

C–N and C–O bond cleavage

–

loss of C8H8 unit(s) H• transfer from H• transfer from terminal position proximal position

– 2H• '' (ab)n

(ab)n

Scheme 4.5. Major dissociation pathways of silverated polystyrene macrocycles, commencing with selective homolytic bond cleavages within the linker substituent. All species are ionized by Ag+ (omitted for brevity). (reproduced with permission from ref. 26).

Hydrogen atom abstraction from a C–H bond by a carboxy radical is exothermic due to the high stability of the COO–H bond.86,87 In the population of fragmenting diradicals, in which the carboxy and benzyl sites are too far apart to permit β-H• transfer between the chain ends, H• rearrangement to the COO• group can take place from the nearest (proximal) benzylic position through an energetically favored six-membered ring transition, as shown in Scheme 4.5; sequential β-H• elimination from the two benzylic radicals generated in this process provides a plausible route to series (ab)n, which is observed 2 m/z units lower than the major series (ab)n''.

42

It is noteworthy that the labile linker blocks the dissociation pathway to (bb)n'' fragments because of selective ring-opening at the linking moiety. The ring-opened isomer lacks the primary radical site needed for a 1,2-phenyl shift, thereby obstructing

(bb)n'' formation.

Despite their structural and stability differences, the two classes of cyclic polystyrenes investigated undergo similar fragmentation chemistry. Both dissociate largely by unzipping, either directly from the [M + Ag]+ precursor ions, or after the loss of linking group elements. More importantly, abundant closed-shell fragment ions in the upper mass region of the MS2 spectrum dominate, in contrast to linear structures, which dissociate to yield abundant open-shell (i.e. radical) ions in the lower mass region of the

MS2 spectrum. These distinctive features allow for conclusive determination of the correct polymer architecture.

4.3. Cyclic Polybutadiene

Our results for polystyrene oligomers reveal that polymer architecture is an important determinant of the fragmentation patterns resulting from free radical chemistry.

MS2 experiments on linear polybutadiene (PB) have shown that also this connectivity favors free radical chemistry when energetically activated.88 Hence, cyclic PB was also examined in order to ascertain whether the preference for efficient unzipping by monomer losses is a general phenomenon of polymers undergoing unimolecular decay through homolytic bond cleavages and radical rearrangements.

43

The cyclic PB was prepared by ring-opening metathesis polymerization of 1,5,9-

79 cyclododecatriene, which generated a polymer with the composition (C4H6)n and no additional functionality (see structure in Figure 4.8). The MALDI-MS2 spectrum of the silverated 17-mer from this product includes dominant fragments due to monomer losses

(depolymerization) in the high and medium mass range along with radical ion fragments in the low mass range, cf. Figure 4.8, as was encountered with the cyclic polystyrenes.

More importantly, the MS2 spectrum of the silverated macrocyclic oligomer is substantially different from the MS2 spectra reported for Ag+ adducts of linear polybutadienes,88 which primarily decompose to low-mass radical ions that include one of the chain end groups (cf. Figure 4.9).

◊ 972

◊ (dd)n" (n = 4-16)

◊

• 918 • J4 ◊ J2 • ◊ J3 ◊ ◊ ◊ ◊

324 ◊ ◊ ◊ ◊ ◊

864

216 809

1025.7

270

755

701

647

323

593 377

485

539 431

300 500 700 900 m/z

Figure 4.8. MALDI-MS2 spectrum of the silverated 17-mer (m/z 1025.7) from a macrocyclic PB obtained by ring-opening metathesis polymerization of 1,5,9- cyclododecatriene.79 (reproduced with permission from ref. 26).

44

CH2–CH2 bond cleavage

(1) loss of ≥ 1 monomer (C H ) units 4 6 (dd) '' (2) intramolecular H• n transfer

Scheme 4.6. Dissociation of silverated polybutadiene macrocycles via homolytic CH2–

CH2 cleavages yielding two allylic radicals at the chain ends. All species are ionized by Ag+. (reproduced with permission from ref. 26).

Scheme 4.6 provides a mechanistic rationalization for the major fragment ion series from the PB macrocycle. The weakest bond in the PB chain is the CH2–CH2 single bond.86,89 Preferential dissociation at such a bond in the Ag+-cationized 17-mer yields an isomeric linear, symmetric diradical with allylic radical sites at both chain ends, cf.

Scheme 4.6. Monomer loss(es) from this intermediate and sequential intramolecular H- atom transfer (disproportionation) between the radical sites leads to fragments with the nominal composition (C4H6)n, bearing a truncated PB chain capped with C4H7

(CH3CH=CHCH2–) and C4H5 (CH2=CHCH=CH–) end groups, as shown in Scheme 4.6.

This series has been termed (dd)''n because its chain ends are created by C–C bond cleavage at the end of a complete repeat unit (“bond d”, cf. Figure 4.9).44

45

• w3 + • J3

w • 2 d • + 2 •

J2

272 216 w 4 1570.2 ^

323 ^ d3 ^ ^ 377 a ^ 13 dn 431 *

* 539 880 485 ^ ^ ^ w

L4 ^ ^ n

647 593 701 + an K 755 ^ ^ 3 * *+ *+ * + + + + ^ ^ + + + + ^ ^ ^ 200 400 600 800 1000 1200 1400 m/z

Figure 4.9. MALDI-MS2 spectrum of the silverated 26-mer from a linear polybutadiene

(PB) prepared by living anionic polymerization in cyclohexane, using sec-C4H9Li for 88 initiation and CH3OH for termination; under these, most favorable conditions for 1,4- 1 addition, ~10% of the monomer is added in 1,2-fashion. Series an, dn, and wn designate • terminal fragment ions, and Jn , Kn, and Ln are internal fragment ions resulting from backbiting rearrangements.88 See Figure 4.10 for the definition of the nomenclature and the structures of the major fragment ion series. (reproduced with permission from ref. 26).

46

w2 x2 y2 z2 w1 x1 y1 z1

a1 b1 c1 d1 a2 b2 c2 d2

dn

wn

an

Figure 4.10. (a) Nomenclature scheme for the MS2 fragment ions from linear 2 polybutadienes and (b) major terminal ions in the MS spectrum of silverated C4H9– 44 + (CH2CH=CHCH2)26–H (the Ag ion is omitted for brevity). Series dn and wn arise from

C–C bond cleavages within repeat units incorporated in 1,4-fashion. Series an result from C–C bond cleavages at units incorporated in 1,2-fashion.88 (reproduced with permission from ref. 26).

• In the low mass region of Figure 4.8, a quite abundant radical ion series, Jn , is detected. These low mass products are attributed to backbiting rearrangements in the allylic diradicals generated after ring opening and monomer loss(es). The observation of a series of radical ions further indicates that the migrating H• may originate from a nearby location (as illustrated in Scheme 4.2 for polystyrene) or from more distant locations down the chain. This behavior is consistent with the lower degree of crowding in PB, as compared to PS, which makes it easier for the reacting sites to approach each other for H• rearrangement.

47

4.4. Conclusions

Linear and cyclic architectures of polystyrenes and polybutadienes show significantly different fragmentation characteristics in tandem mass spectrometry (MS2) experiments. In all cases, dissociation starts with homolytic cleavages in the polymer chain. The incipient radicals generated from linear structures unzip extensively by a combination of monomer losses and backbiting rearrangements; as a result, low-mass dissociation products dominate. The incipient radicals generated from macrocyclic structures, on the other hand, remain in the same molecule and in close proximity. This favors disproportionation, viz. intramolecular H• transfer between the radicals, at the expense of fragmentation via consecutive monomer losses, thus leading to abundant high- mass fragments.

The unique fragmentation reactivities of linear and cyclic polystyrenes and polybutadienes allow for unambiguous determination of the correct architecture.

Architectural assignment is possible by simple visual inspection of relative fragment intensity patterns. Rigorous interpretation of the fragment peaks is, however, essential for identifying specific substituents or linkers in the analyzed molecules. It is worth noting that the molecules investigated decompose via free radical chemistry processes, which lead to fragment ion distributions that reflect the polymer architecture. Other polymers fragmenting via analogous free radical chemistry, such as poly(methyl acrylate)s and poly(methyl methacrylate)s, are expected to show a similar dependence of fragment ion distribution on architecture.

48

CHAPTER V

SEQUENCE ANALYSIS OF STYRENIC COPOLYMERS BY TANDEM MASS

SPECTROMETRY

5.1. Composition and architecture of poly(dimethylsilylstyrene-co-styrene) copolymers.

Figure 5.1a shows the MALDI mass spectrum of the copolymer prepared by reacting 0.0045 mol of initiator (sec-butyllithium, sec-BuLi) with 0.074 mol styrene and

0.0045 mol meta-dimethylsilylstyrene (m-DMSS).74 A bimodal distribution of products is observed (ionized by Na+). The [M+Na]+ ions in the low mass region, centering at ~1500

Da, arise from butyl-initiated, proton-terminated chains of polystyrene homopolymer (A) or poly(m-DMSS-co-styrene) copolymer with one (B) or two (C) m-DMSS comonomer units. Conversely, the [M+Na]+ ions in the high mass region, centering at ~3000 Da, correspond to dimeric (2-armed) architectures with two polymer chains and overall 1-3 m-DMSS units (F-H). Calculated and measured m/z values of select oligomers are included in the expanded spectra of Figures 5.1b-c.

49

(a) A-C F-H

1000 2000 3000 4000 5000 m/z

A A n=14; m=0 n=15; m=0 C 1537.95 calcd. 1537.94 calcd. 1542.01 n=12; m=2 B 1642.02 calcd. 1549.94 n=13; m=1 (b) calcd. 1595.97 1595.98 1549.95

1540 1560 1580 1600 1620 1640 m/z

F F n=26; m=1 H n=27; m=1 calcd. 3004.81 G calcd. 3108.87 n=23; m=3 n=25; m=2 (c) calcd. 3016.80 calcd. 3062.87 3016.96 3004.97 3062.99 3109.05

3020 3040 3060 3080 3100 m/z

Figure 5.1. (a) MALDI mass spectrum of poly(m-dimethylsilylstyrene-co-styrene); all ions are Na+ adducts. (b,c) Expanded views of the (a) low and (b) high mass distribution, containing monomeric products A-C and dimeric (2-armed) products F-H, respectively; in the dimeric product, the m-DMSS comonomers may be located on either of the two arms and arm linking is possible at any of the m-DMSS units. Measured and calculated monoisotopic m/z values are given for the oligomers observed in the expanded views.

50

Figure 5.2. (a) MALDI mass spectrum of poly(p-dimethylsilylstyrene-co-styrene); all ions are Na+ adducts. (b,c) Expanded views of the (a) low and (b) high mass distribution, containing monomeric products A-E and dimeric (2-armed) products F-L, respectively; in the dimeric product, the p-DMSS comonomers may be located on either of the two arms and arm linking is possible at any of the p-DMSS units. See Table 5.1 for the calculated m/z values of the oligomers contained in the expanded views.

51

The copolymer with para-dimethylsilyl styrene (p-DMSS) was prepared by combining 0.0045 mol of sec-BuLi initiator with 0.083 mol styrene and 0.0095 mol p-

DMSS.74 Again, the product distribution is bimodal (Figure 5.2a), although now the proportion of homopolymer (A and L) is lower and the DMSS content of the copolymeric oligomers (B-K) higher due to the larger molar fraction of silylated comonomer.

Calculated and measured m/z values of select [M+Na]+ ions in series A-L are given in

Table 5.1.

52

Table 5.1. Measured vs. calculated monoisotopic m/z values of the oligomers observed in the low (m/z 1490-1630) and high (m/z 3530-3710) mass regions (Figures 5.2b-c) of the MALDI mass spectrum of poly(p-dimethylsilylstyrene-co-styrene).

Series Composition Measured m/z Calculated m/z Δ(m/z) Monomeric oligomers (A-E) a B n = 13; m = 1 1595.93 1595.97 -0.04 D n = 10; m = 3 1607.91 1607.96 -0.05 A n = 15, m = 0 1641.96 1642.01 0.05 C n = 12; m = 2 1653.95 1654.00 -0.05 E n = 9; m = 4 1665.93 1665.99 -0.06 B n = 14, m = 1 1699.98 1700.03 0-.05 D n = 11; m = 3 1711.96 1712.03 -0.07 A n = 16; m = 0 1746.02 1746.01 0.01 C n = 13; m = 2 1758.00 1758.06 -0.06 E n = 10; m = 4 1769.99 1770.05 -0.06 Dimeric (2-armed) oligomers (F-L) a,b H n = 28; m = 3 3537.28 3537.14 0.14 J n = 25; m = 5 3549.26 3549.13 0.13 L n = 33; m = 0 3571.33 3571.20 0.13 G n = 30; m = 2 3583.33 3583.18 0.16 I n = 27; m = 4 3595.32 3595.17 0.15 K n = 24; m = 6 3607.31 3607.15 0.16 F n = 32; m = 1 3629.38 3629.22 0.16 H n = 29; m = 3 3641.36 3641.21 0.15 J n = 26; m = 5 3653.36 3653.19 0.17 L n = 34; m = 0 3675.44 3675.26 0.18 G n = 31; m = 2 3687.42 3687.24 0.18 I n = 28; m = 4 3699.42 3699.23 0.19

a See Figure 5.2a for the monomeric and 2-armed architectures. b Minor products are detected 2 Da below most dimeric distributions, suggesting that further coupling reactions occur, presumably at the reactive silyl hydride groups (currently under investigation).

53

It is noteworthy that the two-armed architecture is observed independent of the

DMSS comonomer used (cf. Figures 5.1-5.2). This byproduct is attributed to a linking reaction involving coupling of radical intermediates and/or nucleophilic displacement of a Si–H hydride by polystyryl lithium, and its exact formation mechanism is still under investigation.74 The focus of this study was to elucidate the comonomer sequence in the two copolymers by MS2. This issue is addressed after a brief discussion of the MS2 characteristics of two reference structures with known sequence, viz. polystyrene terminated by a proton (homopolymer) and polystyrene end-capped with a block of p-

DMSS (block copolymer).

5.2. Reference MS2 spectra of polystyrene and poly(p-DMSS-b-styrene).

The MS2 fragmentation pathways of metal-cationized polystyrene have been thoroughly investigated and are well understood.22,27-28,91,44 Energetically activated

[M+Ag]+ and [M+Li]+ ions of such polymers mainly dissociate through charge-remote free radical chemistry, starting with random homolytic C–C bond cleavages along the backbone to form charged radical intermediates. The latter species decompose to form the observed fragments via typical radical-site reactions, including β bond scissions, backbiting rearrangements, 1,2-phenyl migrations, and hydrogen atom transfer between the incipient radicals created after C–C bond cleavage. For linear polystyrene chains, the predominant fragmentation mode of the initially formed radical intermediates is backbiting through 1,5-hydrogen (“McLafferty”) rearrangement and depolymerization by monomer evaporation through radical induced β C–C bond scissions.27,44 These processes ultimately lead to intense radical ions with one of the original end groups as well as to internal fragments at the low-mass end of the MS2 spectrum; and to two homologous

54 series of fragments with lower intensity in the medium-mass range, each retaining one of the original end groups (α or ω) at one chain end plus a double-bonded methylene group at the other chain end.27,44 Examples of these fragments are provided in Figure 5.3, along with a succinct explanation of the nomenclature used for the types of MS2 fragments observed from linear polystyrenes.44,91

~

~

Figure 5.3. (a) Fragment ion notation for MS2 products from polystyrenes; (b) examples of the radical ion fragments (left) and internal fragments (right) dominating the low-mass region of MS2 spectra; (c) homologous fragment series containing the initiating (α) or terminating (ω) end group and a methylene group at the other chain end. The subscripts give the overall number of complete or partial repeat units and the superscripts the number of DMSS comonomer units.

55

Figure 5.4a depicts the mid range of the MS2 spectrum of the lithiated 17-mer from a polystyrene homopolymer with the connectivity sec-C4H9–(styrene)17–H (m/z

1834.2). This oligomer is present in both poly(DMSS-co-styrene) copolymers investigated. The MS2 spectrum shown was obtained from the homopolymeric component in poly(p-DMSS-co-styrene), but identical spectra result from the homopolymeric component in poly(m-DMSS-co-styrene) as well as from an authentic polystyrene standard with sec-C4H9– (α) and –H (ω) chain ends. Figure 5.4a affirms that the homologous fragment series an and yn dominate the middle mass range of polystyrene

MS2 spectra.

56

y4 + b3" Li y5 (a) C4H9–(styrene)17–H

a4 423.1 y6

377.0 a5 527.2 389.1 a6 y7 a7 y y9 631.2 8 y10 493.2 a8 a9 a10 a11 a

y11 12 y12

597.3

735.3

701.4

943.5

893.4

805.4

909.5

1047.6

1013.6

1117.7

1221.6

1151.6 1255.8

400 600 800 1000 m/z 1200

a5 a4 + b3" Li (b) C4H9–(styrene)11-b-(p-DMSS)2–H 2

y3 493.1

389.2 377.0 419.1 2 a y4 6 435.1 2 2y y5 a 9 2 a7 2 a 10 y10 y6 8 2 a9 2

573.1 y

597.2 y7 8

539.2

643.3

1042.6

701.3

1059.6

805.4

1013.5

747.3

909.4

1163.6

955.5

851.3 1129.2

400 600 800 1000 m/z

Figure 5.4. Mid-mass region of the MALDI-MS2 mass spectra of [M+Li]+ ions from (a) the homopolymeric polystyrene oligomer C4H9–(styrene)17–H (m/z 1834.2) and (b) the copolymeric (end-capped) oligomer with the block connectivity C4H9–(styrene)11-b-(p-

DMSS)2–H (m/z 1534.0). See Figure 5.3c for the structures of an and yn. The b3" fragment (m/z 377) has the connectivity C4H9–(styrene)3–H (the double prime denotes a • saturated chain end, i.e. one more H atom than b3 ).

57

Figure 5.4b depicts the mid range of the MS2 spectrum of a lithiated copolymeric

13-mer, composed of 11 styrene and 2 p-DMSS units. This oligomer was selected from a poly(p-DMSS-b-styrene) block copolymer prepared by end-capping polystyryl lithium with four equivalents of p-DMSS; the corresponding mass spectrum is provided in Figure

5.5. Due to the sequential addition of the comonomers, the p-DMSS block is placed at the terminating (ω) chain end and may be viewed as the ω end group of the polymer chain. As a result, the fragment series an, which is missing the terminating chain end, remains unchanged and is observed at the same m/z values as the an series from the homopolymer, cf. Figures 5.4a and 5.4b. In contract, all y-type fragments in the MS2 spectrum of the copolymeric oligomer are shifted by +116 m/z units, as they contain the p-DMSS block which adds 2 x 58 = 116 Da to their mass (58 Da is the mass difference between the DMSS and styrene repeat units). The y-type ions from the block copolymer

2 have been labeled with the acronym yn (Figure 5.4b), in which the superscript denotes the inclusion of two p-DMSS comonomer units, while the subscript gives the total number of repeat units in the fragment.

58

n=9; m=3 (a) calcd. 1503.9 end-capped dimer 1504.2 end-capped n=18; m=5 calcd. 2818.7 2819.2

1000 1500 2000 2500 3000 3500 4000 m/z (b) n=7; m=5 n=6; m=5 n=8; m=4 calcd. 1619.9 n=9; m=3 calcd. 1515.9 n=10; m=3 calcd. 1561.9 calcd. 1503.9 calcd. 1608.0 n=11; m=2 1504.2 1562.2 1608.3 calcd. 1549.9 n=5; m=6 1620.3 1516.2 n=13; m=1 n=12; m=1 1550.2 calcd. 1573.9 calcd. 1491.9 calcd. 1596.0 1574.2 1492.2 1596.3

1500 1520 1540 1560 1580 1600 m/z

Figure 5.5. (a) MALDI mass spectrum of poly(p-dimethylsilylstyrene-b-styrene), viz. polystyrene end-capped with p-DMSS; all ions are Na+ adducts. (b) Expanded view of the low mass distribution, containing monomeric products with mainly 1-6 p-DMSS repeat units; measured and calculated monoisotopic m/z values are given for the oligomers observed in the displayed m/z range. The dimeric (2-armed) products observed in the high mass distribution contain two H atoms less than the dimeric products from poly(p-dimethylsilylstyrene-co-styrene), which was prepared from a mixture of styrene and p-DMSS (cf. Figure 5.2). A likely structure, generated through linking reactions at two Si–H bonds, is given in Figure 5.5a; such double linking is facilitated by the contiguous arrangement of the p-DMSS units in the end-capped copolymer.

59

It is evident from Figure 5.4 that the an and yn fragment series are contiguous in the middle mass range of the MS2 spectrum and appear at m/z values that reflect the position of the DMSS comonomers in the polymer chain; thus, they can be used to derive sequence information (vide infra). Spectral differences are also observed in the low-mass region of the MS2 spectra (shown in Figures 5.6, 5.8, and 5.10); however, the dominance of internal fragments (which compromise sequence determination) and of very small fragment ions (that do not provide sequence insight) makes the latter region less suitable for the characterization of polymer sequences.

60

Figure 5.6. Low-mass region of MALDI-MS2 the mass spectra of [M+Li]+ ions from (a) the homopolymeric polystyrene oligomer C4H9–(styrene)17–H (m/z 1834.2) and (b) the copolymeric (end-capped) oligomer with the block connectivity C4H9–(styrene)11-b-(p-

DMSS)2–H (m/z 1534.0). See Figure 5.3b for the structures of radicals and closed-shell species.

5.3. Sequence analysis of poly(p-DMSS-co-styrene) and poly(m-DMSS-co-styrene)

The MALDI-ToF tandem mass spectrum in Figure 5.7a is for a lithiated copolymeric 16-mer with 15 styrene and 1 p-DMSS units. This oligomer was selected from the monomeric distribution in the MALDI mass spectrum of poly(p-DMSS-co-

61 styrene) (Figure 5.2a). The synthesis of this copolymer involved simultaneous addition of the two monomers in the reaction vessel followed by addition of the initiator and, after a set reaction time, of the terminating agent. The question to answer is: what is the location of p-DMSS unit in the copolymer chains? The MALDI-MS2 result reveals three different

1 types of fragment ions; these are the an, an (again, the superscript indicates the number of p-DMSS unit and the subscript gives the overall number of p-DMSS plus styrene units) and yn series. Now it is noticed that only fragment series an, which carries the initiating

1 chain end (α) is shifted by +58 Da (indicated by an series in Figure 5.7a), initially partly and later (at higher m/z values) practically completely. A mass increase by 58 Da indicates inclusion of p-DMSS unit in the fragment. In sharp contrast and unlike the end- capped copolymer, the yn series remains largely unchanged (i.e its m/z values are the same as from the homopolymer, cf. Figures 5.4a and 5.7a). MALDI-MS2 data provide evidence that the p-DMSS monomer must be incorporated near the initiator, presumably due to a higher reactivity with polystyryl lithium than styrene.

62

b3"

377.1 (a)

y4

y5 a 423.1 4 y6 1 1 y7 1 1 a4 a5 1a a y a 527.2 7 8 3 a 6 1a y 1a y 1 1a 5 8 9 9 10 a10 y11 11 1 389.2 a

6 631.3 y a7 11

735.4 a8

551.2

447.2

759.4

839.4

655.3

343.1

493.2 967.6

863.5

943.6

1175.8

1071.6

597.3 1047.6 1151.7

701.4 @ 1209.7

805.4 @ @

400 500 600 700 800 900 1000 1100 m/z

b3"

377.1 (b)

2 a4 y 2 2 2 1 2 1 2a 1 2a 4 2 a6 a 1 a8 y9 a9 y10 10 y11 11 y5 a5 7 y8

y6 a4 y

7 897.3

1105.7 1001.5 1 1 1209.6 a3 a a5 a6 y8 4 1 y9

a 1a a7 y 921.5 5 713.3 a 10

6 1 8 817.4

1233.7 1129.6 609.2 1 a y

9 1025.5 11 505.2 a7 a8 # # 400 500 600 700 800 900 1000 1100 m/z

Figure 5.7. Mid-mass region of the MALDI-MS2 mass spectra of [M+Li]+ ions from copolymeric 16-mers of poly(p-DMSS-co-styrene) with the comonomer composition (a)

(styrene)15(p-DMSS)1 (m/z 1788.2) and (b) (styrene)14(p-DMSS)2 (m/z 1846.2). See

Figure 5.3c for the structures of an and yn. The b3" fragment (m/z 377) has the connectivity C4H9–(styrene)3–H (the double prime denotes a saturated chain end, i.e. one • 1 more H atom than b3 ). The sign @ points to trace amounts of yn (n = 8-10); the sign # points to trace amount of an (n = 10-11).

Figure 5.7b shows MALDI-ToF tandem mass spectrum of a lithiated copolymeric

16-mer with 14 styrene and 2 p-DMSS units. Again, this oligomer was selected from the monomeric (linear) distribution in the MALDI mass spectrum of poly(p-DMSS-co- styrene) (Figure 5.2a). It is to be recall that the comonomers were mixed before starting

63

1 the polymerization (vide supra). The fragment ions observed here are series an, yn, an and

2 an series. The spectrum in Figure 5.7b indicates that the yn series mainly stays the same,

1 2 whereas both an and an series are observed in an addition to an series. There is a mass

1 2 increase of 58 Da or 116 Da from series an to an or an, respectively, which is an

1 2 equivalent of one with the inclusion of one (in an) or two p-DMSS units (in an). On the

1 other hand, the proportion of yn ions that shift to yn (i.e they contain one p-DMSS unit)

2 is negligible up to y7 and small for y≥8, and no yn ions are detected above noise level. All these findings are again consistent with the two p-DMSS comonomer units being incorporated near the initiating chain end.

If the comonomer is near the initiator unit, then it should be increasingly present in the larger yn-type fragments. From 16-mers, fragment with up to ~11 repeat units are detectable in the MALDI-MS2 spectra (Figure 5.7). Closer inspection of the fragment patterns reveals that the yn series begins to contain p-DMSS units when it reaches at least

1 1 8 repeat units ( y8 - y11), further confirming that indicating the p-DMSS unit is closer to the initiator group.

The low-mass regions of the MALDI-MS2 spectra of the copolymeric 16-mers from poly(p-DMSS-co-styrene) are shown in Figure 5.8. They are dominated by internal fragments and small terminal radical ions, as was the case for the reference spectra discussed earlier. Both spectra include an ion at m/z 226.1 whose m/z ratio agrees with

1 • • 1 • • b1 , viz. lithiated C4H9-(p-DMSS)1 ; however, in both spectra, b1 is less intense than b1

• (m/z 168.1), viz. C4H9-(styrene)1 . Hence, the p-DMSS unit is not attached directly next to the initiator in the majority of copolymer chains, just somewhere near the initiator unit. It should be mentioned that m/z 226.1 is also present, at a lower relative intensity, in the

64

MS2 spectrum of homopolymeric polystyrene (cf. Figure 5.4a), pointing out that isobaric ions exist at this m/z value. Nevertheless, the intensity ratio [m/z 226.1]/[ m/z 168.1] is much higher for the copolymer, strongly suggesting that m/z 226.1is partly the C4H9-(p-

DMSS)• radical ion (lithiated) in the spectra of the copolymer.

Figure 5.8. Low-mass region of the MALDI-MS2 mass spectra of [M+Li]+ ions from copolymeric 16-mers of poly(p-DMSS-co-styrene) with the comonomer composition (a)

(styrene)15(p-DMSS)1 (m/z 1788.2) and (b) (styrene)14(p-DMSS)2 (m/z 1846.2). See Figure 5.3b for the structures of radicals and closed-shell ions.

65

The MALDI-ToF tandem mass spectrum in Figure 5.9 was acquired from a lithiated copolymeric 14-mer, having 13 styrene and 1 m-DMSS units. This oligomer was selected from the low-mass (monomeric) distribution in the MALDI mass spectrum of poly(m-DMSS-co-styrene) (Figure 5.1a). As with the p-DMSS, the two comonomers were mixed before initiation. The resulting MS2 fragment ions can be divided into four

1 1 different product ion series: an, an, yn and yn. Now, both an and yn fragment ions series

1 1 gradually shift to an and yn as the fragment mass increases (Figure 5.9). Up to 7-8 repeat

1 1 units, the copolymeric fragments ( an, yn) are less abundant than homopolymeric fragments with the same number of repeat units (an, yn); beyond this size, the copolymeric fragments (i.e those including m-DMSS) predominate and become

1 essentially the only sequence ions observed for the largest fragments detected (viz. a11

1 and y11). These MS/MS results are reconciled with a rather random distribution of the m-

DMSS unit within the copolymer chain, suggesting that the meta substituted comonomer and styrene have comparable reactivities.

b3"

377.1 332.1

y4 a4

a5 y5 423.2

389.2 1 1 a a6 y6 1 a3 4 1 1 1 1 y8 1 1 1 1 1 1 338.1 a5 a y7 1 a8 a y 493.2 y 6 a 9 9 y a y y4 527.2 5 1 7 a7 1 a10 10 11 11 y6 y7 1 y y10

a8 y8 a9 9

631.2

597.3

447.2

839.4

343.1

863.5

735.3 551.2

967.5

655.2

585.2

701.3

481.2

759.4

1001.4

1209.7

689.2 1105.7 1175.8

1071.6

793.3

943.4

805.4

909.3

897.4 1047.5

400 500 600 700 800 900 1000 1100 1200 m/z Figure 5.9. Mid-mass range of the MALDI-MS2 mass spectrum of [M+Li]+ from the copolymeric 14-mer of poly(m-DMSS-co-styrene) with the comonomer composition

(styrene)13(m-DMSS)1 (m/z 1580.0). See Figure 5.3c for the structures of an and yn.

66

The low-mass region of the MALDI-MS2 spectrum of the copolymer with one m-

DMSS comonomer unit (Figure 5.10) is very similar with that obtained from the copolymer with one p-DMSS comonomer unit (Figure 5.8a), underscoring that the sequence information rendered by this spectral section is poor. Nevertheless, it is worth

1 • noting that the relative intensity of m/z 226.1, which comprises at least partly b1 fragments, is significantly lower with a m-DMSS comonomer which is not attached near the initiator group.

• b1 • •

z2 & J2

168.1 202.1 y2 & K2 K1 y3 & K3 b • • 111.1 2

z1 a3

319.1

272.1

258.1

98.1 215.1

226.1

285.1

161.1 306.1

100 150 200 250 300 m/z Figure 5.10. Low-mass range of the MALDI-MS2 mass spectrum of [M+Li]+ from the copolymeric 14-mer of poly(m-DMSS-co-styrene) with the comonomer composition

(styrene)13(m-DMSS)1 (m/z 1580.0). See Figure 5.3b for the structures of radicals and closed-shell species.

67

5.4. Conclusions

Copolymers of functionalized styrene and styrene were successfully sequenced based on their fragmentation characteristics resulting from MALDI-ToF-MS2 experiments. The p-DMSS MS2 data reveal a tapered sequence, with the comonomer near the initiating chain end. On the other hand, the m-DMSS MS2 result indicates a randomly distributed comonomer a long the polymer chain. Furthermore, MALDI-ToF-MS2 could easily differentiate between a copolymer produced by end-capping and one produced by mixing of comonomers.

This study showed for the first time that isomeric polymer sequences can not only be differentiated by 2D mass spectrometry, but also elucidated to determine the specific or preferred locations of comonomers. An important prerequisite is, however, that reference spectra are available and fragmentation pathways well understood.

68

CHAPTER VI

MALDI-TOF/TOF TANDEM MASS SPECTROMETRY OF LINEAR IN-CHAIN

SUBSTITUTED, CYCLIC WITH TWO LINKER UNITS, AND FOUR-ARM STAR-

BRANCHED POLYSTYRENES

6.1. Linear in-chain substituted PS

The linear in-chain functionalized PS was prepared by living anionic polymerization. First, 22.5 mmol of styrene monomer and 1.11 mmol of pent-4-en-1- yllithium, supplied in ampules, were combined inside a dry box to produce polystyryllithium; after the addition of 0.44 mmol of dichlorodimethylsilane (linking agent) telechelic in-chain substituted polystyrene was formed (Scheme 6.1). The

MALDI-MS spectrum (Figure 6.1) of this polymer shows a narrow molecular weight distribution, centered around m/z 4400 with expected styrene repeat unit (104 Da). The inset shows an expanded view of the m/z region between the 31-mer and 32-mer. The ion observed at m/z 3529.16 agrees very well with a silverated 31-mer with the structure of the desired linear in-chain substituted PS (calculated m/z 3529.01).

69

Scheme 6.1. Reaction scheme for the linear in-chain substituted PS and the PS macrocycle with two linker units analyzed.

70

3529.16

3540 3560 3580 3600 3620 3640 m/z

2000 2500 3000 3500 4000 4500 5000 5500 6000 m/z

Figure 6.1. MALDI-ToF mass spectrum of linear in-chain substituted PS. A zoom view of the region between the 31-mer and 32-mer is given in the upper right corner. All ions are [M + Ag]+ adducts.

The MALDI-MS2 spectrum of the silverated 31-mer from the linear in-chain substituted PS includes three sizable fragment ion distributions, which have been labeled with the symbols &, +, and $ (Figure 6.2). This fragmentation pattern is dramatically different from those of chain-end substituted or macrocycles PS, which were discussed in chapter IV, and very similar to that for other centrally substituted polystyrenes.44,88 The presence of approximately Gaussian fragment distributions in MS2 spectra is characteristic for in-chain (centrally) functionalized PS and can be used to diagnose such architectures.

α The bn and bni series are reconciled by initial bond cleavage at the Si-C bonds

(Scheme 6.2) which should be weak because of overcrowding near the Si atom.44 The Si- centered and benzylic radials emerging this way can undergo consecutive H• loss to yield

71 bni and bn fragments, respectively, which contains one original end group (C5H9) plus an unsaturated moiety as the second end group (Scheme 6.2). A double bond to silicon (as in

90 bni) is unlikely in solution, but can exist in the gas phase. Alternatively, the Si-centered

• radical may undergo backbiting before H loss to form an isomeric bni fragment with a

CH=CHPh instead of a Si=CH2 double bond. The remaining sizable series an and the

α β minor series anb, on the other hand, are accounted for by C -C bond cleavage which yields two incipient radicals, one benzylic next to the linker and one primary without the linker unit. The primary radical can further decompose by losing monomer and H• or Ph• radicals to form series an and anb, respectively (Scheme 6.2). Backbiting in the benzylic radical next to Si, on the other hand, leads to internal fragment K3i (m/z 463) smaller an fragments (cf. Scheme 4.2).

In addition to these dissociations that occur at or near the in-chain substituent, random hemolytic C-C bond cleavages along the PS backbone can take place. These were

• • • • discussed in chapter IV and are the primary source of fragments b1 , b2 , b3 , J2 , and K3 which dominate the low-mass region of the MS2 spectrum; in addition, the random

2 dissociations contribute to the an series in the medium mass range of the MS spectrum

(see chapter V).

72

•

b1 279.61

(a) 3529.16

• b2 & an series without Si linker K3 + bn series without Si linker •

J2 418.65 $ bni series with Si linker

&

383.68 & & & • b3 & $

1645.18 $ $ 1749.23 & 1541.14 &$ $ $

K3i 133.81 & $ 1437.07

$ 3487.90 & $

487.68 & $

192.29 & & $ 1333.00

& 2001.43

2105.40 $ 3423.38

& 2209.35 $ 3320.42

1228.95 $ 1897.12 $

2314.37 $

2417.16

1124.85

1793.28

2521.18

604.68 1020.82

1691.16 &

708.71

812.75 2624.75

916.76 +

+ + 2728.90 2832.48 + + + 2935.91 1585.21 &

+ & 3039.99 3144.16 + + + 3216.76 500 1000 1500 2000 2500 3000 3500 m/z

& an series without Si linker a 15 + bn series without Si linker a a16 14 & $ b series with Si linker & & ni

1645.23 a17 1749.20 (b) 1541.13 &

1853.28 a18 b17i b16i & $ $

b15i 1957.32

$ 2001.35 1897.02 b14i b b16 $ 15 + b + 1793.28 b17 b13 14 +

+ b13i +

1839.25 1689.13 $ 1735.19 a 1943.32

1631.22 16b 1527.09 a 17b a a19b 1584.99 18b

a15b 1673.02

1777.14

1985.47 1882.33

1500 1550 1600 1650 1700 1750 1800 1850 1900 1950 2000 m/z

Figure 6.2. (a) MALDI-MS2 spectrum of the silverated 31-mer (m/z 3529.2) from the linear in-chain substituted PS shown as inset and in Scheme 6.1. (b) Expanded view of the m/z 1500-2000 region. See scheme 6.2 for the structures of an, anb, bn, and bni.

73

(2) H• or Ph• loss (to a & a ) α β n nb

(3) Backbiting (to an & K3i)

α β

α (2) H• loss

Scheme 6.2. Major fragmentation pathways of linear in-chain substituted PS.

6.2 Cyclic PS with two linker units

The cyclic with two linker units was prepared from the linear in-chain percursor

(Scheme 6.1) by cyclization with Grubb’s first generation catalyst, viz. bis(tricyclohexylphosphine)benzylidene ruthenium(IV) chloride,81 as discussed in chapter

IV. The MALDI mass spectrum (Figure 6.3) of this macrocycle shows one narrow molecular weight distribution with the expected 104-Da repeat unit. The silverated n- mers of the cyclic polymer appear 28 Da lower than the same n-mers from the linear in- chain precursor and this is consistent with Grubbs metathesis mechanism. The zoomed

MALDI-MS spectrum (Figure 6.3) shows the m/z region between the 31-mer and 32-mer

74 of this PS macrocycle. The calculated and measured m/z of the 31-mer are 3500.98 Da

and 3501.23 Da, respectively. No impurities or byproducts are detected.

3605.32 3501.23

3500 3520 3540 3560 3580 3600 m/z

2000 2500 3000 3500 4000 4500 5000 5500 6000 m/z

Figure 6.3. MALDI-ToF mass spectrum of the macrocyclic PS with two linker units prepared as shown in Scheme 6.1. A zoomed view of the m/z region containing the 31- mer and 32-mer is given in the upper right corner. All ions are [M + Ag]+ adducts.

75

The Figure 6.4 shows the MALDI-MS2 spectrum of the 31-mer of this cyclic polystyrene. As described in section 6.1 for linear in-chain substituted PS, breakup of the

Si-Cα and Cα-Cβ bonds is facile due to sterical crowding and silicon’s ability to stabilize

C-centered radicals in α position. These bond cleavages produce charged diradicals which, as discussed in chapter IV of this dissertation, can fragment further via typical free radical chemistry. Monomer evaporation and consecutive intramolecular H• transfer from one radical by the other (disproportionation) gives rise to the isomeric series (bb)ne″ and

α α β (ab)ni″, resulting after initial Si-C or C -C bond cleavage, respectively (cf. Scheme 6.3, left side). The subscripted e and i indicate whether the Si linker is an end group (e) or an internal substituent (i) in the final fragment. Monomer evaporation accompanied by 1,2- phenyl shift or loss of the linker (Scheme 6.3, right side) gives rise to the other two prominent fragment series, viz. (bb)ni″ and (ab)n″, respectively. The overall fragmentation pattern is more complex than that encountered for macrocycles with only one linker (cf.

Figures 6.4 and 4.5), but nonetheless includes the features diagnostic for the microcyclic architecture, namely dominant closed-shell ions in the upper mass region of the MS2 spectrum.

76

3471.03

† (bb)ne″ & (ab)ni″ † 3501.22

‡ (bb)ni″ † 3397.41

(bb)n″ 3293.51

† † ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ ‡ 420.48 ‡

301.47

3189.52

3085.61

192.18

2239.91

2135.04 2343.82

2029.19

2446.81

2551.70

2656.21

2759.70

1925.74

2863.26

2966.89

357.49 1821.96 89.42 † † † ‡ 1568.91 ‡

1464.86 †

1360.71 1717.94

1256.69 † † † † ‡ ‡

1152.57

1048.62 944.53

500 1000 1500 2000 2500 3000 3500 m/z

Figure 6.4. MALDI-MS2 spectrum of the silverated 31-mer (m/z 3501.2) from the macrocyle with two linker units shown as inset and in Scheme 6.1. The subscripts e and i denote inclusion of the Si linker at the chain end (e) or in an internal location (i), respectively (cf. Scheme 6.3).

77

† (bb)ne″ (ab)n″

(1) - (C8H8)x (2) - (C H ) (1) - 8 8 x (2) Intramolecular (3) Intramolecular H• transfer H• transfer

(1) 1,2-phenyl shift (1) - (C8H8)x (2) Loss of CH2=CH-CH2-Ph + (C8H8)x (2) Intramolecular (3) Intramolecular H• transfer H• transfer

(bb)ni″ ‡ † (ab) ″ ni

Scheme 6.3. Major dissociation pathways of the silverated PS macrocycles with two linkers, one inert (C8H14) and one with weak bonds in α and β positions to a Si atom. Random homolytic cleavages within the PS chains create diradicals that can ultimately yield the same types of fragments as those generated after initial Cα-Cβ bond cleavage.

78

6.3. Four-arm star-branched polystyrene

The 4-arm star-branched PS (Scheme 6.4) was prepared by reaction of polystyryllithium with 1,2-bis(dichloro(methyl)silyl)ethane and subsequent addition of oxirane before termination acidic methanol. The oxirane converts unreacted polystyryllithium hydroxyethyl-terminated polystyrene, which can be separated from the non polar star product by thin-layer chromatography. The MALDI mass spectrum of the

4-arm star-branched polystyrene (Figure 6.5) shows two narrow molecular weight distributions, one in the low-mass and one in the high-mass region, centering at about m/z

1200 and 4400,respectively. The distribution in the low-mass region corresponds to unreacted PSLi that was not functionalized with oxirane during the synthesis and is composed of polystyrene chains having a sec-butyl group at the initiating and a proton at the terminating chain-end. The calculated and measured mass-to-ratio of the 9-mer in this low-mass distribution are 1101.547 and 1101.642, respectively. The molecular weight distribution in the high-mass region of Figure 6.5, on the other hand, is the expected 4- arm star-branched PS. The calculated and measured m/z of the 32-mer the latter product are 3779.222 and 3779.485, respectively.

79

(2) (1)

Scheme 6.4. Reaction sequence to the 4-arm star-branched PS analyzed.

80

1205.712

1101.642

3883.578 3779.485

3780 3800 3820 3840 3860 3880 m/z 1100 1120 1140 1160 1180 1200 m/z

1500 2000 2500 3000 3500 4000 4500 5000 5500 6000 m/z

Figure 6.5. MALDI-ToF mass spectrum of the 4-arm star-branched PS produced from a tetrachlorosilane according to reaction sequence in Scheme 6.4. . Zoom in low-mass range upper in the left is between 9-mer and 10-mer and in high-mass region upper in the right is between 32-mer and 33-mer, respectively. Zoomed views of select m/z regions in the low-mass and high-mass distributions are given in the upper left or upper right corners, respectively. All ions ar [M + Ag]+ adducts.

The MALDI tandem mass spectra of two 4-arm star-branched polystyrene n-mers are depicted in Figures 6.6 and 6.8. Both include three distributions stretching from the mid-mass to the high-mass region which are approximately one arm apart from each other. Fragmentation begins with cleavage of the weakest bonds in the polymer; in this case, most labile must be the Si-Cα bonds (see Scheme 6.5). Fragmentation can start at any of these bonds. Comparison of the tandem mass spectra in Figures 6.6 and 6.8 with the tandem mass spectrum of linear PS on page 27 of this dissertation reveals significant differences. For example, series an and yn which normally dominate the mid-mass range

81 of linear PS are absent in the MS2 spectra of the 32-mer and 33-mer of the 4-arm star- branched PS. Furthermore, the tandem mass spectra of the 4-arm star-branched polystyrene contain three distributions which are not present in the MALDI-MS2 spectrum of linear PS. Based on this information, we can easily distinguish a linear from a star-branched architecture. Meanwhile, the number of fragment distributions observed reflects the number of detachable arms and, hence, identifies the number of arms in the analyzed star polymer.

3779.508

3737.186 160.001

2-arm PS 115.564 192.197 linear PS 3-arm PS

ECG = 56 Da ECG = 226 Da ECG = 284 Da

371.661

2104.086

2000.833

994.621

1098.647

267.518

1896.003

2207.817

1202.655

3671.180

890.631

2312.102

1792.785

1307.684

2993.339

2889.511

3569.596

786.628

3096.934

316.610

2785.490

2416.087

420.620

1411.695

1688.825

2681.945

3200.918

2519.634

712.571

1514.785

3304.276

1584.769

3407.639 592.599

500 1000 1500 2000 2500 3000 3500 m/z

Figure 6.6. MALDI-MS2 spectrum of the silverated 32-mer from the 4-arm star-branched PS shown as inset and in Scheme 6.4 (m/z 3779.49). The total masses of end and central groups are marked on top of the 3 fragment distributions observed.

82

#

# (a)

1098.647 994.621 # 104 Da

890.631 + 30 Da

920.540

1024.586

1128.591

904.573

1112.607

1008.601

1142.620

934.591

1038.611 1082.619

900 925 950 975 1000 1025 1050 1075 1100 1125 1150 m/z

*

* 2104.086

2000.833 (b) *

* 1896.003

104 Da 2207.817

- 44 Da

+ 90 Da

2194.790

2090.822

1986.737

2164.808

2060.858

1956.931

1882.680

1852.899

2046.911

2149.965

1837.905

1826.798

1929.944

1941.177

2137.758 2034.459

1850 1900 1950 2000 2050 2100 2150 2200 m/z

Figure 6.7. Zoomed MALDI-MS2 spectra of the silverated 32-mer from the star- branched PS shown in Figure 6.6, showing the m/z region of the (a) linaer and (b) 2- armed PS fragments. The main series in these regions are marked by # and *, respectively, and correspond to the linear PS and 2-arm PS fragments shown in Scheme 6.5.

83

159.983

3883.572

192.180

3841.432

115.534 371.712

Linear PS 2-arm PS

3-arm PS

267.538

2104.114

3774.484

2208.267

2000.739

996.652

1204.678

1098.686

2311.980

1308.726

890.652

1896.791

418.678

2416.691

2993.209

2889.315

1791.825

3097.852

786.652

1411.740

3201.456

2519.949

2785.397

3672.640

682.654

1514.840

3305.214

1688.917

2681.677 3407.529

500 1000 1500 2000 2500 3000 3500 m/z

Figure 6.8. MALDI-MS2 spectrum of the silverated 33-mer from the 4-arm star-branched PS shown as inset and in Scheme 6.4 (m/z 3883.58).

84

3-arm PS

4-arm PS

2-arm PS Linear PS

Scheme 6.5. Major fragmentation pathways for a star-branched PS with 4 arms attached to Si atom. These reactions involve arm eliminations, commencing with Si-Cα bond cleavage.

85

•

+

(1) •

(2) H• transfer

- H•

bn

+

3-arm bn

2-arm bn - 44

2-arm bn bn + 30

Scheme 6.6. Charge-remote free radical chemistry accounting for all major MS2 fragments formed from silverated 4-arm star-branched polystyrene oligomers. The second PS arm may be cleaved from the same Si site as the first (shown here) or from the Si site still containing 2 PS arms (as shown in Scheme 6.5). The former pathway leads to a –Si(CH3)H2 terminal group which reconciles more readily the minor bn + 30 and 2-arm bn - 44 fragments. The minor 2-arm fragment possessing an extra 90-Da moiety (see text) nd α β arises by cleavage of the 2 arm at the C -C bond, giving rise to a –Si(CH3)(H)CH2Ph terminal group which is 90 Da heavier than –Si(CH3)H2.

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6.4 Conclusions

Each of the three PS architectures considered in this chapter produce unique fragment ion series and distinctive fragment ion intensity distributions, which permit their conclusive differentiation both from each as well as from linear, chain-end functionalized

PS chains.

All three polystyrenes fragment via charge-remote free radical chemistry, commencing primarily via Si-Cα bond cleavage and, to a lesser extent, via (Si) Cα-Cβ bond cleavage. The type of linker and polymer architecture determine the consecutive reactions following these initial bond cleavages and, hence, also the ultimately observed fragments.

It is interesting to note that this study reports the first MS2 spectra for singly charged polymer ions > m/z 3000. The largest precursor ion examined, viz. the silverated

4-arm star-branched 33-mer, has 597 atoms and 1785 degrees of freedom. Its efficient fragmentation within the microsecond time window of MALDI-ToF/ToF mass spectrometers (Figure 6.8) is attributed to the facile cleavage of Si-C bonds and to combined collisionally activated dissociation and photodissociation at the increased laser power used in the MALDI-MS2 experiments.

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CHAPTER VII

SUMMARY

The work presented in this dissertation confirmed MALDI tandem mass spectrometry as a powerful tool for synthetic polymer analysis. Chapter IV of this dissertation showed that linear and cyclic architectures of polystyrenes and polybutadienes generate significantly different fragmentation characteristics in twodimensional mass spectrometry (MS2) experiments. For all linear and cyclic polymers studied, dissociation starts with homolytic cleavages in the polymer chain. The incipient radicals generated from linear structures unzip extensively by a combination of monomer losses and backbiting rearrangements; as a result, low-mass dissociation products are more intense. The incipient radicals generated from macrocyclic structures, remain in the same molecule and in close proximity. This favors disproportionation, viz. intramolecular H• transfer between the radicals, at the expense of fragmentation via consecutive monomer losses, thus leading to abundant high-mass fragments. Finally, for centrally substituted and more complex star-branched polymers, homolytic dissociation primarily occurs at the linking sites, yielding fragment distributions that reveal the size distribution of the linked polymer chains.

88

The unique fragmentation reactivities of linear, cyclic, and branched polystyrenes and polybutadienes allow for unambiguous determination of the correct architecture.

Architectural assignment is possible by simple visual inspection of relative fragment intensity patterns without rigorous data interpretation. It is noteworthy that all molecules investigated mainly decompose via free radical chemistry processes, which generally cause no (or less) molecular isomerization than charge-induced processes and, thus, generate fragment ion distributions that reflect the polymer architecture.

In chapter V, tandem mass spectrometry was used to sequence copolymers of substituted styrene and styrene based on the fragments observed in MALDI-MS2 spectra, in comparison to the fragments formed from homopolymer and copolymer standards. The tandem mass spectra of the copolymer with p-DMSS revealed a block-like sequence and this suggest that p-DMSS is slightly more reactive than styrene. The MALDI-MS2 spectra of the copolymer with m-DMSS indicated that a random sequence was formed and this is consistent with m-DMSS and styrene having comparable reactivities. The sequencing strategy used here should be helpful in future determinations of comonomer location in other types of copolymeric species, provided that the tandem mass spectrometry characteristics of the corresponding homopolymers are well understood.

Lastly, chapter VI described the characterization of linear in-chain substituted, cyclic with two linker units, and 4-arm star-branched PS polymers by multidimensional mass spectrometry. Again, by inspecting the mid-mass and high-mass ranges of the tandem mass spectra obtained from these molecules, it was possible to assign the correct architecture based on differences observed in the fragments formed and their intensity distributions.

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Dr. Michael Schubert

President, Bruker Daltonics, Inc. Life Sciences Division

Managing Director / Geschäftsführer, Bruker Daltonik GmbH

Fahrenheitstr. 4, D-28359 Bremen

Tel.: +49 421 2205 0

Email: [email protected]

Von: Aleer M. Yol [mailto:[email protected]] Gesendet: Donnerstag, 30. Mai 2013 23:19 An: Schubert Michael Betreff: Copyright Permission

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