Introduction

Star polymers are branched macromolecules consisting of several linear polymeric chains radiating from a relatively small central core [1,2,3]. The distinctive topology of star polymers provides multiple functional groups and leads to lower hydrodynamic volume, lower viscosity, and lower degree of aggregation in solution as compared to linear polymers with similar molecular weight [1, 4,5,6,7]. The unique chemical and physical properties resulting from such features make star polymers particularly suitable for a variety of applications, including catalysis [8], imaging [9, 10], coatings [11], and drug/gene delivery [1, 12,13,14,15,16]. The properties of star-branched polymers are strongly affected by the chemical structure of the central core and each arm, degree of branching, and individual arm length [3, 4]. Consequently, characterization of the number of branches and the length of each branch is essential to develop star polymers with optimal physicochemical properties for the desired application.

Two appropriate mass spectrometry (MS) methods for the determination of architectural and topology features of synthetic polymers are tandem mass spectrometry (MS2) [17] and ion mobility mass spectrometry (IM-MS) [17,18,19,20], which reveal microstructure detail by probing the fragmentation chemistry or size/shape of the analyzed sample, respectively. IM-MS studies on branched polyethers [21] and glycopolymers [22] have documented that multiple charging (≥ 2) is needed to cause visible differences in the 3-D sizes/shapes of the branched polymer vs. a linear polymer with similar molecular weight. This prerequisite restricts the use of IM-MS to polymers that can be ionized by electrospray ionization (ESI) and are large enough to support multiple charges. For smaller oligomers and prepolymers (Mn < 8000 Da), MS2 could be more suitable as this method can provide sequence and connectivity insight for singly charged sample ions [17, 23].

MS2 experiments via collisionally activated dissociation (CAD) or laser-induced fragmentation (LIFT) [24] have been performed in positive ion mode on polymers with linear [25,26,27,28,29,30,31], cyclic [32,33,34,35,36,37], branched [22, 38], and dendritic [39] architectures to identify their primary structures. Previous MS2 studies on branched polymers aimed at proving the presence of branched architecture(s) by detecting fragments that were impossible to form by isomeric linear structures [22, 38]. The present work investigates for the first time whether MS2 can be employed to ascertain specific, important structural features of star-branched polyethers, namely their number of branches and the corresponding branch lengths. The polymers were ionized by matrix-assisted laser desorption ionization (MALDI) which is widely employed in polymer laboratories and typically generates singly charged ions. As will be shown in this study, arms emanating from a central, functionalized core are readily cleaved under MALDI-MS2 conditions, thus revealing the number of branches and the individual arm chain lengths.

Experimental

Mass Spectrometry

MALDI-MS and MALDI-MS2 spectra were measured on a Bruker UltraFlex-III MALDI-ToF/ToF mass spectrometer (Bruker Daltonics, Billerica, MA) equipped with a Nd: YAG laser (355 nm). Solutions of the matrix, trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene] malononitrile (DCTB, Alfa Aesar, 99 + %), sodium trifluoroacetate cationizing salt (NaTFA, Aldrich, 98%), and the star polymers were prepared in tetrahydrofuran (THF, Aldrich, 99.9%) at concentrations of 20 mg/mL, 10 mg/mL, and 10 mg/mL, respectively. Matrix and salt were mixed in the ratio 10:1 (v/v). About 0.5 μL of matrix/salt mixture was deposited onto a sample spot of the 384-well ground steel target plate and allowed to dry under ambient conditions. About 0.5 μL of the star polymer solution was then deposited on top of the dried matrix/salt, followed by another 0.5 μL of matrix/salt mixture onto the dried polymer. The sample spots were completely dry before the target plate was introduced into the mass spectrometer. This three-layer sandwich sample preparation protocol led to the formation of abundant [M + Na]+ ions from the star polymers. The mass-to-charge ratio (m/z) scale was calibrated by poly(methyl methacrylate) (PMMA) standards with a molecular weight close to the expected mass of the samples.

The ToF/ToF instrument used consists of a short linear ToF analyzer coupled to a reflectron ToF tube; this configuration permits both MALDI-MS as well as MALDI-MS2 analyses. For single-stage MALDI-MS spectra, both ToF devices are used as a single-reflectron ToF mass spectrometer. MALDI-MS2 spectra are acquired using the LIFT mode [24, 40]. It involves the use of (1) increased laser intensity to activate the precursor ions so that they undergo post-source decay (PSD) in the linear ToF region; (2) isolation of the desired precursor ion and its fragments, which move with the same velocity, by a timed ion selector (TIS); and (3) post-acceleration of the isolated ions for mass analysis in the reflectron ToF region [24, 40].

ESI-MS2 experiments were performed on a Waters Synapt quadrupole/time-of-flight (Q/ToF) mass spectrometer (Waters, Milford, MA) at the settings described in the supporting information. Solutions of the star polymers in THF (0.1 mg/mL) were directly injected into the ESI source with a syringe pump at a flow rate of 10 μL/min. Precursor ions were mass-selected by Q and induced to fragment via collisionally activated dissociation (CAD) in the trap cell located between the Q and ToF analyzers.

Polymers

A set of high purity homo-arm and mikto-arm poly(ethylene glycol) (PEG) star polymers were synthesized by a stepwise “graft onto” method [4, 41] utilizing epoxide ring-opening and copper-catalyzed azide-alkyne cycloaddition (CuAAC) “click” chemistry [42] to attach monomethoxy-terminated linear PEG chains with an average molecular weight (Mn) of 2 kDa or 750 Da (Sigma-Aldrich, St. Louis, MO) to the same core. The PEG star polymers prepared by this approach are shown in Figure 1 and include a 2-arm mikto-star (2-kDa and 750-Da PEG chains), a 2-arm homo-star (2-kDa PEG chains), and a 3-arm homo-star (2-kDa PEG chains). The step-by-step synthesis of such architectures has been reported in a previous publication [43]. The raw synthetic products were purified by vacuum filtration, precipitation in cold hexane, and further purification by size exclusion chromatography on a Bio-Beads resin (Bio-Rad Laboratories, Hercules, CA) [43].

Figure 1
figure 1

Structures of (a) 2-arm mikto-star (2-kDa and 750-Da PEG arms), (b) 2-arm homo-star (2-kDa PEG arms), and (c) 3-arm homo-star (2-kDa arms) investigated. (d) Nomenclature scheme for the fragments from a linear PEG with methoxy end groups, which is also applied to the star-branched PEG polymers (all contain methoxy chain ends). The bond cleavages create a new end group which is usually a carbonyl (for cn) or vinyl functionality (for bn); see also text

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Results and Discussion

The MALDI-MS spectra of the star-branched polymers show single and narrow molecular weight distributions in the expected molecular weight ranges (cf. Fig. S1). The m/z values of the observed oligomers in each spectrum correspond to [M + Na]+ ions with the desired core functionality and methyl groups at all chain ends [43]. MALDI-MS confirms the exceptional purity and composition of the star polymers but provides no insight on their architectures.

The MALDI-MS2 spectra of select oligomers near the average molecular weight of the 2-arm mikto-star (2- and 0.75-kDa PEG arms), 2-arm homo-star (2-kDa PEG arms), and 3-arm homo-star (2-kDa PEG arms) are depicted in Figure 2; all were acquired in positive ion mode using the LIFT technique. Two major fragment distributions are observed for the 2-arm mikto-star (Figure 2a), one centered at m/z ~ 750 and the other at m/z ~ 2000. This result is in excellent agreement with the proposed structure which was synthesized using one short (750 Da) and one long (2 kDa) PEG arm. Conversely, the 2-arm homo-star prepared from 2-kDa PEG arms gives rise to only one major fragment distribution, centered at m/z ~ 2000 (Figure 2b), consistent with having two equally sized arms in this case. Meanwhile, two major fragment distributions are observed for the 3-arm homo-star with 2-kDa arms, one centered at m/z ~ 2000 and the other at m/z ~ 4000, corresponding to fragments that contain one arm and two arms, respectively. The MALDI-MS2 data of Figure 2 indicate that bond cleavages occur preferentially at the core-branch joining points. The released arms are observed at the molecular weights of the original polymer chain reactants, thus allowing for the determination of the identity and length of the arms. Furthermore, the number of fragment distributions, combined with their average masses, reveals the number of arms and degree of branching. Fairly similar fragmentation patterns are obtained by ESI-MS2 (cf. Fig. S2). It is worth noting, however, that ESI produces molecular ions in many different charge states and that ESI-MS2 experiments on the doubly or more highly charged precursor ions generates fragment distributions varying in mass and charge, thus making spectral analysis and architectural assignments more challenging. In contrast, MALDI-MS2 leads to spectra with markedly less complexity that are easier to interpret.

Figure 2
figure 2

MALDI-MS2 spectra of the [M + Na]+ ions from the (a) 63-mer of the 2-arm mikto-star (m/z 2997.8), (b) 90-mer of the 2-arm homo-star (m/z 4186.5), and (c) 131-mer of the 3-arm homo-star (m/z 6163.7); m/z values are labeled on top of select ions

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Bond cleavages at or near the core-branch joining points could be favored for several reasons. The release of steric strain and the reduction of crowding near the core could contribute to the preference of such dissociations, especially for the more highly branched architecture. The main cause of such reactivity is presumably the stabilization provided by the core to the ionic or radical intermediates traversed during fragmentation. Indeed, all major fragment series formed upon MS2 are reconciled by charge-induced or charge-remote dissociations at or near the triazole ring, as discussed for the three stars examined in the following sections.

2-Arm Mikto-Star (2-kDa and 750-Da PEG Arms)

Metal-cationized polyether ions generally dissociate via both charge-induced as well as charge-remote reactions that cleave the ether bonds [23, 44]. The predominant fragmentation mode is determined by the size of the dissociating ion and the degree of activation. Earlier studies have indicated that short PEG chains dissociate by either mechanism, with charge-induced dissociations favored at low internal energies (and vice versa), whereas longer PEGs (~ 2000 Da and beyond) largely fragment through charge-remote processes [23, 45]. Both types of these chains are present in the 2-arm mikto-star. Further, either of the two carbon-oxygen bonds in the C–O–C ether functionality can be cleaved, and each one may lead to different fragments for asymmetrically substituted PEG chains like the 2-arm mikto-star; for differentiation, these two types of bond cleavages will be referred to as C–O and O–C bond dissociation. Both of these dissociations generate two products: one including the short chain (m repeat units) and the other the long chain (n repeat units); one of these products will also contain the internal linking substituent (indicated by a subscripted letter (i)). Note that all fragments contain one CH3O– chain end from the PEG reactants used to synthesize the stars. Fragmentation creates a new chain end (cf. series bn and cn in Figure 1); these newly created chain ends can be unsaturated (bn or cn) or saturated in which case they are designated with a double prime (for example, cn″).

Charge-induced dissociation near the core-branch juncture of the short arm accounts for two of the homologous fragment series observed, viz. cm″ from the short arm (Figure 3a) and bni from the long arm (Figure 3b). Scheme 1 presents a mechanistic rationalization for this reaction pathway, which is promoted by the Lewis acid character of the Na+ charge. Na+ coordination at the O atom of a C–O bond weakens this bond, facilitating heterolytic cleavage. An unstable primary carbocation and a sodium alkoxylate emerge from this bond cleavage, which can rearrange via an energetically favorable 1,2-hydride transfer to a more stable ion-dipole complex between a secondary alkoxyethyl cation and a sodium alkoxylate. Proton transfer from the carbocation to the negatively charged alkoxylate, accompanied by Na+ ligand rearrangement, produces a Na+-bound complex of the separated chain segments, which can dissociate to yield fragment cm″ (short chain with saturated chain end) or bni (long chain with linker and vinyl chain end). In a linear unsubstituted PEG, such C–O bond scission can take place anywhere along the chain [23, 45]. In the triazole functionalized 2-arm mikto-star, this dissociation occurs preferably on the short arm next to the triazole linker, where the nascent primary carbocation emerging after C–O bond dissociation can be stabilized by electrostatic interaction (intramolecular solvation) with a basic triazole nitrogen atom, as shown in Scheme 1 (note that the hydroxy group on the long arm is most likely hydrogen-bonded to the nearby triazole nitrogen atom, thereby obstructing a similar neighboring group effect in the long arm).

Figure 3
figure 3

Expanded views of the (a) lower and (b) higher mass regions in the MALDI-MS2 spectrum of the sodiated 63-mer of the 2-arm mikto-star (m/z 2997.8), showing the fragments containing the (a) short and (b) long PEG arm, respectively; m/z values are labeled on top of select peaks. See text and Schemes 1, 2, 3, and 4 and S1-S2 for the structures and nomenclature of the fragment series observed; a subscripted letter (i) indicates the inclusion of the internal linking substituent

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Scheme 1
scheme 1

Charge-induced dissociation of a C–O bond in the short arm of the sodiated 2-arm mikto-star (2-kDa and 0.75-kDa PEG arms), generating fragments with (bni) or without (cm″) the linking substituent; rH− and rH+ designate hydride and proton transfer, respectively. The n and m subscripts refer to fragments containing the long PEG chain or short PEG chain, respectively. The subscripted letter (i) denotes the inclusion of the internal linker and the superscripted double prime symbol (″) denotes a newly formed saturated chain end (the hydroxy group of cm″)

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The unique substitution pattern at the long arm of the mikto-star enables a different charge-induced fragmentation mode that detaches the entire long arm at the triazole junction to create fragment series Sm″ (Figure 3a) and Ln (Figure 3b) comprising the complete short arm or long arm, respectively (cf. Scheme 2). Here, heterolytic C–N bond cleavage catalyzed by the Na+ Lewis acid and concomitant 1,2-hydride shift produce a tertiary carbocation interacting with an amide anion. Proton abstraction from the carbocation by the amide nitrogen and H+/Na+ exchange between the detaching segments gives rise to the mentioned fragment ions. The oxygen-rich long arm is detected in sodiated form, whereas the nitrogen-rich (more basic) short arm is observed protonated. The newly formed end groups of homologous series Sm″ and Ln confirm the core-branch connectivity of the 2-arm mikto-star.

Scheme 2
scheme 2

Charge-induced dissociation of the C–N bond in the long arm of the sodiated 2-arm mikto-star (2-kDa and 0.75-kDa PEG arms), generating fragments that contain parts of the linking substituent and either the long (Ln) or short (Sm″) PEG chain; rH−, rH+, and rH+/Na+ designate hydride transfer, proton transfer, and H+/Na+ exchange, respectively

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The reactions discussed compete with charge-remote fragmentations [46], which are most prevalent in longer polyether chains, where the metal ion is well sequestered by several ether oxygens and cannot easily be mobilized to the reactive site to induce heterolytic bond cleavage. Several of the observed fragment series are reconciled by charge-remote homolytic ether bond cleavages which, again, are preferred near the core-branch junction where newly formed radicals can be stabilized by the adjacent triazole ring. Scheme 3a illustrates such a pathway for the O–C bond nearest to the triazole junction on the long arm. Homolytic scission of this bond creates two incipient radical ions, viz. cn• and bmi•. Consecutive H• loss from cn• gives rise to series cn comprising the long arm and an aldehyde end group (dominant fragments in Figure 3b). Interestingly, the complementary series, bmi, formed by H• loss from, bmi•, is not observed; this is attributed to the proximity of the triazole ring, which enables rearrangement of the incipient bmi• radical ions to more stable isomeric species that expel the linker to yield cm• and, by consecutive H• loss, series cm comprising the short arm and an aldehyde end group and appearing 2 m/z units below series cm″ (Figure 3a). A similar charge-remote pathway initiated by cleavage of the C-O bond nearest to the triazole junction on the long arm (Schemes 3b and S1) leads to fragment series bn and cm (Figures 3b and 3a, respectively).

Scheme 3
scheme 3

Charge-remote homolytic (a) O–C and (b) C–O bond dissociation in the long arm of the 2-arm mikto-star (2-kDa and 0.75-kDa PEG chains), generating fragments with a terminal double bond and containing either the long arm (cn and bn) or the short arm (cm). See Scheme S1 for the detailed pathway to the bn + cm fragments originating via a similar mechanism from C–O bond dissociation

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Charge-remote homolytic ether bond cleavages also take place in the short arm (Schemes 4 and S2). Once more, dissociation of bonds proximate to the core-branch junction is favored due to the stabilization the triazole ring can provide to one of the radicals arising upon ether bond scission. Because of the different substitution pattern on the short arm side, the rearranged radical ions do not expel the linker in this case, but rather lose an H• radical to form a double bond at the newly created end group. The overall reaction sequence commences with homolysis of the O–C or C–O bond near the triazole ring and ultimately gives rise to fragment series bm + cni (Scheme 4) or fragment series cm + bni (Scheme S2), respectively.

Scheme 4
scheme 4

Charge-remote homolytic O–C bond dissociation in the short arm of the 2-arm mikto-star (2-kDa and 0.75-kDa PEG chains), generating fragments with a double bond in their chain ends and containing either the short arm (bm) or the long arm plus the linker as end group (cni)

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It is worth noting that the MALDI-MS2 spectrum of the 2-arm mikto-star (Figure 2a) is dramatically different from the MALDI-MS2 spectra reported for linear PEG architectures, which show fragments from random ether bond cleavages across the entire chain and stretching from the molecular ion region down to m/z < 200 [23]. For comparative purpose, the MALDI-MS2 spectrum of the sodiated 47-mer from linear CH3O–PEG–OH (m/z 2124.3) is shown in Fig. S3. Linear PEGs and star-branched congeners with a triazole core can readily be differentiated based on the distinct fragmentation patterns in Figure 2a vs. Fig. S3.

2-Arm Homo-Star (2-kDa PEG Arms)

The 2-arm homo-star generates only one major distribution upon MALDI-MS2 (cf. Figures 2b and 4), as the two PEG arms of this polymer have the same size. The two arms are, however, linked differently to the triazole core: one is attached to a triazole carbon atom, similar to the short arm of the mikto-star, while the other is connected to a triazole nitrogen atom through a hydroxy-substituted bridge, like the long arm of the mikto-star. As a consequence, the homo-star undergoes the same types of dissociations encountered with the corresponding mikto-star, including charge-induced fragmentation via the pathways of Scheme 1 (to cn″) and Scheme 2 (to Ln″ (the same connectivity as Sm″ in Scheme 2) as well as charge-remote fragmentation via the pathways of Scheme 3 (to cn + bn) and Scheme 4 (to bn + cni), cf. Figure 4.

Figure 4
figure 4

Expanded view of the m/z 1600–2400 region in the MALDI-MS2 spectrum of the sodiated 90-mer of the 2-arm homo-star (m/z 4186.5), showing the dominant fragment distributions containing one PEG arm; m/z values are labeled on top of select peaks

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3-Arm Homo-Star (2-kDa PEG Arms)

Two major fragment distributions are present in the MALDI-MS2 spectrum of the 3-arm homo-star, centered at around m/z 2000 and 4000 and arising from fragment ions with one and two 2-kDa PEG arms, respectively. The fragments in the m/z ~ 2000 region include (in decreasing relative intensity) series cn″, cn, bn, and Ln″ (Fig. S4a); this fragmentation pattern is similar with that obtained from the 2-arm homo-star (Figure 4). The dominant series in the m/z ~ 4000 region (Fig. S4b) contain the 2-arm fragment Dn/Dn* which is co-produced with Ln″ by charge-induced N–C bond cleavage, as rationalized in Scheme 5. This fragment is observed both in the protonated form (Dn), due to the inclusion of a basic triazole moiety, as well as in sodiated form (Dn*), due to the presence of two long polyether chains. The reduction of crowding brought upon losing one arm must contribute to the high yield of fragments Dn/Dn*. It is noteworthy that the 3-arm homo-star dissociates efficiently upon MALDI-MS2 in spite of its high mass [47].

Scheme 5
scheme 5

Charge-induced dissociation of the N–C bond in the long arm closest to the hydroxy substituent of the sodiated 3-arm homo-star (2-kDa arms), generating fragments that contain one (Ln″) or two (Dn and Dn*) long PEG chains; rH−, rH+, and rH+/Na+ designate hydride transfer, proton transfer, and H+/Na+ exchange, respectively

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Conclusions

This study documented the utility of tandem mass spectrometry for the topological and architectural differentiation and characterization of star-branched polyethers synthesized using alkyne-azide click chemistry, which creates triazole linkages. The polyethers investigated encompass 2-arm mikto, 2-arm homo-, and 3-arm homo-architectures. Single-stage MALDI-MS confirmed the high purity and narrow molecular weight distribution of the analyzed samples. MALDI-MS2 experiments further demonstrated that activation of the sodiated star polymers preferentially causes bond scissions near the triazole core-branch joining points due mainly to energetically favorable heterolytic and homolytic bond cleavages near the triazole ring and, to some extent, to release of steric strain and/or reduction of crowding as well. These factors result in the detachment of complete arms from the core to generate fragment ions at the expected molecular weight of each arm. The number of fragment distributions observed combined with the m/z values of the fragment ions permit conclusive assignment of the degree of branching and branch lengths. Analogous findings have been reported for polystyrene-based stars with arms emanating from a Si-containing core which can stabilize radical intermediates [17, 48]. The corroborating results of our study allow to project that MS2 will be a generally useful means to determine star architecture if the arms are attached to a functional core that can stabilize ionic or radical intermediates.