Electrospray Ionisation Tandem Mass
Spectrometry of Poly [(R,S)-3-hydroxybutanoic
Acid] Telechelics Containing Primary Hydroxy
End Groups
Hu¨lya Arslan1†, Graz˙yna Adamus2, Baki Hazer1,3and Marek Kowalczuk2* 1Zonguldak Karaelmas University, Department of Chemistry, 67100 Zonguldak, Turkey
2Polish Academy of Sciences, Centre for Polymer Chemistry, 41-800 Zabrze, Poland
3TUBITAK-Marmara Research Center, Food Science and Technologies Research Institute, Gebze, 41470 Kocaeli, Turkey
Evaluation of polymer end-capping reactions with the aid of electrospray ionisation tandem mass spectrometry techniques (ESI-MSn) allows characterisation of novel poly[(R,S)-3-hydroxybutanoic acid] (a-PHB) telechelics, containing primary hydroxyl groups at both polymer chain ends. The chemical structures of individual mass-selected macromolecules of the well-defined a-PHB telechelics have been defined in this way, and fragmentation mechanisms have been proposed. Copyright# 1999 John Wiley & Sons, Ltd.
Received 27 August 1999; Revised 12 October 1999; Accepted 13 October 1999
The term ‘telechelics’ is derived from the Greek words tele (end) and chelos (claw), and refers to oligomers possessing identical reactive groups at each chain end of a linear macromolecule. Such functionalised species may be prepared predominantly by anionic, cationic and group transfer polymerisation (GTP).1 Anionic ring-opening polymerisation of b-butyrolactone leads to ‘living’ oligo-mers and polyoligo-mers, analogues of natural ones,2and several novel functionalised derivatives of poly(3-hydroxybutanoic acid) (PHB) and its copolymers can be synthesised on this way.3Stereochemical control of this process is also possible under specific reaction conditions.4,5 Among aliphatic polyesters, PHB as well as other polyhydroxyalkanoates (PHAs) constitute an interesting group of polymers due to their susceptibility to hydrolytic and enzymatic biodegra-dation.6,7 They have recently been extensively studied via mass spectrometric separation of the individual pseudo-molecular ions formed by ‘soft’ ionisation techniques (including fast-atom bombardment (FAB), desorption chemical ionisation (DCI) matrix-assisted laser desorption ionisation time-of-flight (MALDI-TOF) atmospheric pres-sure chemical ionisation (APCI) and electrospray ionisation (ESI)) in order to characterise the sequence distribution of naturally occurring PHA copolymers,8,9 end groups of synthetic PHB analogues,10 as well as the oligomeric products of their biodegradation.11,12
The ESI-MSnfragmentation analysis of individual chains of aliphatic polyesters, in particular those of poly(3-hydroxybutanoic acid) containing 3-hydroxybutanoate and crotonate end groups, has been reported by some of us recently. Using this particular technique, determination of molecular masses and structures of fragment ions of
mass-selected macromolecules has been accomplished, thus exhibiting the chemical nature of a polymer and its end groups.10In the present study, we report for the first time the application of ESI-MSn techniques for the evaluation of polymer end-capping reactions and characterisation of novel poly[(R,S)-3-hydroxybutanoic acid] telechelics con-taining primary hydroxyl groups at both polymer chain ends.
EXPERIMENTAL a-PHB telechelics
Poly[(R,S)-3-hydroxybutanoic acid] telechelics containing primary hydroxyl groups at both polymer chain ends were synthesised by anionic ring-opening polymerisation of (R,S)-b-butyrolactone with 4-hydroxybutanoic acid sodium salt/ 18-crown-6 complex as an initiator, followed by termination of polymerisation with bromodecanol to form polymer 2a containing a hydroxydecyl ester end group, and with bromoethanol to form polymer 2b with a hydroxyethyl ester end group (see Table 1, Scheme 1). The telechelics 2 selected for ESI-MSnexperiments (Table 1) were addition-ally characterised by 1H and 13C NMR, gel permeation chromatography (GPC) and Fourier transform infrared (FT-IR)spectroscopy.
Table 1. Samples of a-PHB telechelics analysed by ESI-MSn
Sample no. Alkylating agent
Polymera
(Scheme 1) Yield [%] Mnb Mw/Mnb
1 HO(CH2)10Br 2a 81 1200 1.2
2 HO(CH2)2Br 2b 82 1100 1.1
aThe molar ratio of (R,S)-b-butyrolactone to 4-hydroxybutanoic acid sodium salt/ 18-crown-6 complex was equal to: [Mo]/[Io] = 9; solvent THF. b Mn: number-average molecular mass; Mw/Mn: polydispersity index, estimated by GPC experiments.
*Correspondence to: M. Kowalczuk, Polish Academy of Sciences, Centre for Polymer Chemistry, 41-800 Zabrze, Poland.
†On temporary leave to the Centre of Polymer Chemistry, Polish Acadamy of Sciences under the framework of a TUBITAK research grant.
# 1999 John Wiley & Sons, Ltd.
RAPID COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom. 13, 2433–2438 (1999)
The global picture of the polymers 2 is provided by the
1
H-NMR analysis, which shows characteristic signals of the protons corresponding to the poly(3-hydroxybutanoic acid) chain and primary alcohol end groups (Fig. 1).
In addition to NMR, analysis was performed using ESI-MS in order to determine the molecular masses of the individual polymer macromolecules and verify the chemical homogeneity of the polyesters 2 obtained after end-capping reactions.
GPC analysis
Number-average molecular mass (Mn) and polydispersity index (Mw/Mn) were estimated by GPC experiments conducted in THF solution at 35°C, at a flow rate of 1 mL/min using a Spectra- Physics 8800 solvent delivery system with Plgel 3mm MIXED-E ultrahigh efficiency
column and a Shodex SE 61 refractive index detector. Polystyrene standards with low polydispersity were used to generate a calibration curve.
NMR analysis
The NMR spectra were recorded using a Varian VCR-300 multinuclear spectrometer. The1H NMR spectra were run in CDCl3 using tetramethylsilane (TMS) as an internal
standard.
ESI-MSnexperiments
Electrospray tandem mass spectrometric analysis (ESI-MSn) was performed with a Finnigan LCQ ion trap mass spectrometer (Finnigan, San Jose, CA, USA). The polymer sample was dissolved in methanol (1.0 mg/mL) and solution Scheme 1.
Figure 1.
Rapid Commun. Mass Spectrom. 13, 2433–2438 (1999) Copyright# 1999 John Wiley & Sons, Ltd.
was introduced into the ESI source by continuous infusion by means of the instrument syringe pump at a rate of 3mL/ min. The ESI source was operated at 4.25 kV and the capillary heater was set to 200°C.
For ESI-MSn experiments, mass-selected monoisotopic molecular adduct ions were isolated in the ion trap and collisionally activated with 32% ejection RF-amplitude at standard He pressure. The experiments were performed in positive-ion mode.
RESULTS AND DISCUSSION
Analysis by ESI-MS was performed on poly[R,S)-3-hydroxybutanoic acid] telechelics 2, containing primary hydroxy groups at both polymer chain ends (Samples 1 and 2, Table 1), obtained after the end-capping reaction of polymer 1 with selected n-bromo alcohols (Scheme 1).
A comparison of the positive-ion ESI mass spectra of poly(3-hydroxybutanoic acid) telechelics 2a and 2b (Fig. 2) revealed the presence of the corresponding sets of sodium adduct ions of macromolecules containing one 4-hydro-xybutanoate end group and hydroxydecyl (Fig. 2(a)) or hydroxyethyl (Fig. 2(b)) ester groups on the second polymer chain end, respectively. The most abundant sodium adduct ion in the ESI mass spectrum of polymer 2a was located at m/z 1143.5 (Fig. 2(a)), and was assigned to the decamer [HO(CH2)3ÿC(O)O[CH(CH3)CH2C(O)O]10(CH2)10OH
Na](1143.3 Da). The corresponding sodium adduct ion of the decamer containing a hydroxyethyl ester end group was located at m/z 1031.5 (Fig. 2(b)). The difference between the m/z values of the sodium adduct ions of the macromolecules
2a and 2b. which have the same degree of polymerisation but different end groups, is 112 Da, corresponding to the molecular mass difference of eight CH2units between the
corresponding end-capping moieties. Respective values of number-average molecular mass Mn(ESI) equal to 1130 for polymer 2a and 1090 for polymer 2b as well as polydispersity index Mw/Mn(ESI) equal to 1.05 for polymer 2a and 1.09 for polymer 2b were estimated.12 The estimated values are comparable with those determined by GPC experiments (Table 1).
The ESI mass spectrometric analysis of polymers 2 (Fig. 2) also indicated minor peaks corresponding to a small residual amount of polymer 1 in each sample (1.3% in the case of Sample 1 and 2.6% in the case of Sample 2), which was not detectable by1H NMR (Fig. 1). Comparison of Figs 2(a) and 2(b) shows clearly that the series of minor peaks is not dependent on the termination agent employed and it was
assigned to the residual amount of sodium adduct ions of polymer 1. The most abundant sodium adduct ion in the ESI mass spectrum of polymer 1 was located at m/z 1009.1 and it was still observed in the ESI mass spectra of polymers 2 (Fig. 2). This ion was assigned to the decamer [HO(CH2)3ÿC(O)O[CH(CH3)CH2C(O)O]9CH(CH3)CH2
-C(O)ONa Na] (1009.2 Da). Due to the coincidence between molecular masses of sodium adduct ions of polymer 1 and respective values calculated for the hypothetical protonated form of polymer 2b the correctness of the above assignment was also confirmed by ESI-MSn experiments. It was thus demonstrated that, due to the high sensitivity and specificity of ESI-MS, the end-capping reaction of poly(3-hydroxybutanoic acid) can be easily followed by this technique and the chemical homogeneity of the resulting telechelics may be verified in this way. ESI-MSnfragmentation experiments
The investigation of the fragmentation mechanism of a-PHB mass-selected ions (carried out in positive-ion mode) demonstrated that, in MS2experiments, during fragmenta-tion of parent ions of poly(3-hydroxybutanoic acid) macro-molecules bearing carboxylate and 3-hydroxybutanoate end groups, two sets of fragment ions are formed due to primary expulsion of crotonic acid (86 Da) and 3-hydroxybutanoic acid (104 Da). The fragmentation of the ions of related a-PHB macromolecules which contain crotonate instead of 3-hydroxybutanoate end groups generates only one set of fragment ions formed by loss of crotonic acid from both polymer chain ends.10
The ESI-MSn fragmentation experiments on mass-selected ions of telechelics 2 revealed fragmentation pathways from both ends of individual macromolecule ions. The MS2spectrum of the sodium adduct ion of the nonamer of 2a (m/z 1057) is presented in Fig. 3(a). The fragment ion at m/z 953 is formed by the loss of 4-hydroxybutanoic acid (104 Da). On the other hand, the fragment ion at m/z 815 is formed by the expulsion of 10-hydroxydecyl crotonate (242 Da). The MS2 fragmentation of the parent ion of the decamer of 2b (m/z 1031) also shows fragmentation at both ends of the polymer molecule (Fig. 3(b)). The fragment ion at m/z 927 is formed by the loss of 4-hydroxybutanoic acid (104 Da), and the fragment ion at m/z 901 is formed by expulsion of 2-hydroxyethyl crotonate (130 Da).
MS3spectra of the sodium adduct ion of the decamer 2b (m/z 1031) are presented in Figs 4(a) and 4(b) and the Figure 2.
corresponding fragmentation mechanism is proposed in Scheme 2. In the MS3experiment the further fragmentation of the ion at m/z 927 (containing crotonate and hydroxy ester extremities), and that of the ion at m/z 901 (containing
4-hydroxybutanoate and carboxylate extremities), leads to two sets of fragment ions (Figs 4 (a), (b); Scheme 2, MS3). However, the fragment ion at m/z 797 (containing crotonate and carboxylate end groups), formed in both of the above Figure 3.
Rapid Commun. Mass Spectrom. 13, 2433–2438 (1999) Copyright# 1999 John Wiley & Sons, Ltd.
MS3experiments, creates in the MS4experiment only one set of fragment ions corresponding to successive losses of crotonic acid (Fig. 4(c); Scheme 2, MS4).
The results of the ESI-MSnexperiments, illustrated with
these examples of individual mass-selected molecular ions, demonstrate that the polymers 2 contain macromol-ecular chains with a hydroxy group at each end of the expected structure. The fragmentation of sodium adduct Figure 4.
ions of a-PHB telechelics proceeds via both polymer chain ends, with the primary expulsion of 4-hydroxbuta-noic acid and crotonic hydroxy ester molecules followed by the successive losses of crotonic acid. The fragment ions thus formed after several MSnexperiments form the respective a-PHB fragment ions containing crotonate and carboxylic extremities, and its further fragmentation leads to only one characteristic set of fragment ions (Fig. 4(c); MS4).
CONCLUSIONS
The characterisation of novel a-PHB telechelics has been accomplished with the aid of analysis by ESI-MS, and the convenience of this technique for evaluation of polymer end-capping reactions has been demonstrated. The im-plementation of multistep mass spectrometry significantly improves the analysis of the detailed structure of individual chains in a-PHB bearing primary hydroxy end groups, thus demonstrating the general utility of the ESI-MSn technique in studies of these biologically related macromolecules.
Acknowledgements
This work was partially supported by a EUREKA E! 2004 ‘MICROPOL’ Grant.
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Scheme 2.
Rapid Commun. Mass Spectrom. 13, 2433–2438 (1999) Copyright# 1999 John Wiley & Sons, Ltd.
