Forbidden Chemistry: Two-Photon Pathway in [2+2] Cycloaddition of

Jul 6, 2017 - Two-photon excitation provides high spatial resolution in three dimensions of the corresponding chemical or physical processes, allowing...
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Forbidden Chemistry: Two-Photon Pathway in [2+2] Cycloaddition of Maleimides Mikhail V. Tsurkan,*,† Christiane Jungnickel,†,§ Michael Schlierf,§ and Carsten Werner‡,†,§ †

Max Bergmann Center of Biomaterials, Leibniz Institute of Polymer Research, Hohe Strasse 6, 01069 Dresden, Germany Center for Regenerative Therapies Dresden, Technische Universität Dresden, Fetscherstraße 105, 01307 Dresden, Germany § B CUBE - Center for Molecular Bioengineering, Technische Universität Dresden, Arnoldstraße 18, 01307 Dresden, Germany ‡

S Supporting Information *

unwanted side products or require photoinitiators, such approaches enable creation and functionalization of extracellular matrix-like hydrogel materials.3 The development of photoinitiator-free two-photon-induced coupling reactions could advance the field of biomaterials and regenerative medicine by providing two-photon click chemistry for the spatially controlled creation of materials under mild physiological conditions, making stereolithography and 3D printing for the creation of de novo engineered living tissues possible. Maleimide derivatives undergo [2+2] cycloaddition when excited with UV light14 even in the absence of a photoinitiator, which makes this reaction attractive for biological applications. The maximum quantum yield of photoreactions in N-substituted maleimides is red-shifted from its absorption maximum,15,16 and longer-wavelength light (350−370 nm) is used to study their reactions14 (Figure 1A). The excitation of 10% aqueous solution

ABSTRACT: Two-photon excitation provides high spatial resolution in three dimensions of the corresponding chemical or physical processes, allowing submicrometer structuring in stereolithography and three-dimensional (3D) microfabrication. While studying two-photon structuring applications, we observed an undescribed phenomenon in photochemistry that dictates reactivity of maleimide groups in two-photon mode. A low-absorbance transition formerly ignored in classical photochemistry has been found for maleimides. This transition was assigned to symmetry-breaking donor−acceptor complex formation, which revealed a formally forbidden pathway in [2+2] cycloaddition reactions of maleimide moieties. This synthetic pathway allowed for the creation of hydrogel materials under physiological conditions at low laser excitation energy (0.1 J/cm2 at 800 nm) without the use of photoinitiators, which makes it truly two-photon click chemistry.

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io-orthogonal click chemistry reactions, such as Michael addition, the Diels−Alder reaction, and azide−alkyne cycloaddition, are used for synthesis of bulk hydrogels for biological and medical applications.1,2 However, their usefulness for biomaterial structuring is limited because they do not provide spatial control of their reactivity. As an alternative to click chemistry, light-catalyzed reactions are useful in material formation and structuring.3,4 Light as a reactant can be spatially specific because advanced optic devices enable light to be focused in an area of several dozen nanometers. Light-induced polymerization in biomaterial fabrication is promising in three-dimensional (3D) printing5 and stereolithography.6 Nevertheless, such progress is limited because formation of biomaterials in situ with living matter requires efficient reactivity under mild physiological conditions, where the majority of suitable photosensitive chemicals are insoluble7 or unreactive under the required biocompatible wavelength window (700−1200 nm). Two-photon processes in which two photons act as one with double energy are possible solutions, providing precise spatial control and an effective reactivity in the visible-wavelength window.7 Works utilizing two-photon approaches for the structuring and functionalization of various materials have been focused on two-photon photolysis8−10 and photoinitiatorcatalyzed reactions.7,11,12 Although these reactions result in © 2017 American Chemical Society

Figure 1. Spatially controlled formation of PEG-Mal4 hydrogel structures through two-photon reactions. (A) Reaction scheme of PEG-Mal4 hydrogel formation; (B) images of hydrogel fine structures formed with 800 nm laser excitation in (a) water, (b) PBS and (c) cell culture medium; (C) scanning electron microscopy images of hydrogel fine structures formed on maleimide-functionalized surfaces;13 (D) examination of two-photon hydrogel formation under different wavelengths and laser power at a writing speed of 15 μm/s. Received: May 8, 2017 Published: July 6, 2017 10184

DOI: 10.1021/jacs.7b04484 J. Am. Chem. Soc. 2017, 139, 10184−10187

Communication

Journal of the American Chemical Society

sulfoxide or DMF. The irradiation of the sample with UV light (255 or 366 nm) for 24 h led to a high yield of the [2+2] cyclobutyl cycloaddition product (Figure 2B,C), whereas similar to the PEG-Mal4 conjugate, no reaction was observed after extensive temperature treatment (90 °C, ∼4 days) (Figure 2E). The irradiation of the model compound with a 405 nm laser (continuous wave, 200 mW) for 10 h revealed the formation of approximately 5% of the same [2+2] cyclobutyl cycloaddition product, as shown by HPLC and ESI-MS (Figure S16, 17) and as characterized by NMR (Figure S30). The reactivity of maleimides at 405 nm indicates the possibility of the [2+2] cycloaddition reaction in two-photon mode at 800 nm. It required 4 days of sample irradiation (because of ∼2 × 10−9 mL excitation volume) in two-photon mode (800 nm, 100 mW, 100 fs, 80 MHz pulsed laser) to register product formation. The product was barely detectable by its absorbance in HPLC analysis but was clearly observed in the ESI-MS spectrum (Figure 2D). The high similarity of the products’ ESI-MS spectra in one- and two- photon experiments allowed us to conclude the identical structure of these compounds. Together, these experiments revealed the two-photon reaction at 800 nm is the [2+2] cycloaddition, which raises questions about the mechanism of this reaction. Back calculation of the one-photon process led to a wavelength of 400 nm, where no significant absorption of maleimide has been reported (Figure 3A). Accurate spectral measurements of

of four-armed maleimide-terminated polyethylene glycol (PEGMal4, Mw = 10 kDa) for 30 min with 366 nm light resulted in stable hydrogel network formation, whereas the corresponding use of 720−740 nm light in a two-photon laser setup (Figure S1) did not lead to hydrogel formation, even at 500 mW light intensity (the peak power density was 6.25 × 109 W in 12 × 10−10 cm2 and the pulse energy was 6.25 × 10−3 J). The absence of twophoton reactivity was expected because of the low absorbance (ε ≈ 10 L·(mol·cm)−1 at 360 nm) of the n−π* transition in maleimides, responsible for the [2+2] cycloaddition reaction.15,16 By accident, the PEG-Mal4 sample was treated in a two-photon instrument with an 800 nm laser, which resulted in formation of a distinct hydrogel structure (Figure 1B,C and S8−S10). Further exploration revealed it is reproducible for various maleimide conjugates that could react with each other in 3D structuring.17 Although these experiments revealed the high efficiency of maleimide two-photon coupling under 800 nm excitation, the nature of this reaction remained a mystery because no absorbance of the maleimide moiety at 400 nm had been reported, contradicting classical photochemistry. In the present work, we investigated the mechanism of maleimide reactivity in two-photon mode. Temperature treatments of the PEG-Mal4 sample (90 °C, >8 h) did not lead to hydrogel formation. A detailed analysis of hydrogel formation revealed its efficiency within a region of 780−840 nm with a maximum at approximately 800 nm (Figure 1D and S3−S7). To elucidate the nature of the observed reaction, we investigated the reactivity of a model compound, N-succinimidyl 3-maleimidopropionate (Mal-NHS) (Figure 2A), the synthetic precursor to the PEG-Mal4 conjugate (Figure S11). The model compound allowed NMR, HPLC, and mass spectrometry to be used for the structural characterization of the reaction product, which otherwise could not be revealed in the PEG-Mal4 conjugate reaction. Mal-NHS is not soluble in aqueous solvents, and all model compound reactions were carried out in dimethyl

Figure 3. Electronic spectra of PEG-Mal4 conjugate. (A) UV−vis spectrum of the maleimide; the two-photon excitation area in which the [2+2] cycloaddition reaction is effective is highlighted; (B) linear dependence of the absorbance at 290 nm on the sample concentration; (C) linear dependence of the absorbance at 400 nm on the sample concentration; (D) emission spectrum changes in response to the excitation wavelength, where the dashed spectra correspond to the increase and the solid spectra to the decrease of the fluorescence with increasing excitation wavelength; (E) solvatochromic shift of the emission and (F) the excitation spectra.

PEG-Mal4 revealed maleimide groups exhibit a weak absorption at approximately 400 nm dependent on concentration of the maleimide moiety (ε ≈ 1 L mol−1 cm−1) (Figure 3B,C), similar to the π−π* (290 nm, ε ≈ 500 L mol−1 cm−1) transition used to determine the concentration of maleimides. The absorbance at 400 nm could be considered as a broadened UV peak; however, it could also be a peak shoulder fitted as a distinct peak (Figure S21). Investigation of such weak absorbance is difficult because it approaches the sensitivity limit of UV instruments, but could be characterized indirectly.18 We noticed the model compound had a distinct visible fluorescence when irradiated with 405 nm light (Figure 2B). Because this fluorescence must be associated with the 400 nm transition, we investigated it. The emission spectra of the PEG-Mal4 conjugate show a fluorescence maximum at 505 nm for the 360−440 nm excitation wavelength range, whereas in accordance with reports,16 almost no fluorescence was observed for the π−π* band excitation at

Figure 2. Model compound reactivity. (A) Reaction scheme of Nsuccinimidyl 3-maleimidopropionate [2+2] cycloaddition; (B) image of sample fluorescence under 405 nm excitation; (C) HPLC and ESI-MS analyses of the model compound treated with 366 nm light; (D) HPLC and ESI-MS analyses of the model compound treated for 4 days with 800 nm laser light; (E) HPLC and ESI-MS analyses of the model compound heated (90 °C) in the dark for 4 days. 10185

DOI: 10.1021/jacs.7b04484 J. Am. Chem. Soc. 2017, 139, 10184−10187

Communication

Journal of the American Chemical Society 290 nm, and only a weak fluorescence (420 nm) was observed for the n−π* band at 360 nm (Figure 3D). The excitation spectra show the maximum fluorescence was reached when the sample was irradiated at 400 nm (Figure 3D−F). These results reveal the weak absorbance at 400 nm is not an artifact and is the source of PEG-Mal4 fluorescence. Thus, the weak absorbance of the maleimide samples at 400 nm represents the electronic transition that defines the maleimide reactivity in two-photon mode (800 nm), where the excitation of the n−π* (360 nm) transition that is 10 times stronger has been found inactive. The electronic spectra of maleimide and its derivatives have previously been studied, and the observed weak band at 400 nm does not belong to the maleimide electronic transitions.15,16 The transition at 400 nm can be assigned to the formation of a donor−acceptor π−π association or donor−acceptor complex (DAC), well-known for various heterogeneous π-conjugated compounds.19 Such DACs are noncovalent complexes with delocalized electron density and are held together by π-stacking. The DAC formation results in orbital coupling, which lowers the energy of the HOMO−LUMO transition (Figure 4A) and can be

known; for example, electron transfer (charge separation) has been described for homogeneous molecules, and its mechanism can be explained as a disproportionate redox reaction.22 Using this analogy, we hypothesize the maleimide DAC forms through symmetry breaking disproportionate to the donor and acceptor (Figure 4B). The overlap of the HOMO of the donor and LUMO of the acceptor can be considered the main force for the heterogeneous DAC formation (Figure 4A). Homogenous DAC would therefore have no obvious energy benefits; the formation of such complexes is unlikely because the reduced entropy disfavors complex formation. Solvation energy could favor DAC formation in polar solvents because of the complex polarization. In this case, the solvation energy should decrease the energy of the CT excited state of the DAC and the energy required for this transition. Hence, the CT transition should be red-shifted from the maleimide n−π* transition (360 nm) and therefore should be observed. This hypothesis can be tested because the wavelength of such CT transitions is proportional to polarity of the solvent and therefore should be solvatochromic.23 Solvatochromism of the PEG-Mal4 DAC charge-transfer transition is shown in Figure 3E,F, where a nearly 50 nm red shift in the emission is observed between the dichloromethane and water solutions. The observed transition at 400 nm is the CT transition of the symmetry-breaking DAC of maleimide groups. The PEG-(Mal)4 polymer is a centrosymmetric molecule; therefore, the excitation, allowed in one-photon absorption (400 nm), is forbidden in two-photon absorption and should be shifted to a lower wavelength (higher energy) compared to the observed 800 nm.23−25 However, the two-photon polymerization (Figure 1D) peaks at 800 nm, supporting the hypothesis the chromophore is not PEG-(Mal)4 but the symmetry-breaking DAC of maleimide moieties is not centrosymmetric. Although the nature of the mysterious transition at 400 nm appears to be clear, its contribution to the mechanism of hydrogel formation must be defined. The originally proposed triplet-driven mechanism of [2+2] cycloaddition26 is disfavored, and the charge transfer mechanism is considered the dominant reaction pathway (Figure 4C).27 This understanding led to a new breakthrough in [2+2] cycloaddition catalysis that resulted in an enhanced reaction rate as well as precise stereoselectivity control28,29 even in the reaction of chemically inert olefins.30 For PEG-Mal4 hydrogel formation, according to this mechanism, maleimide groups, after excitation, must diffuse to their counterpart molecule for the charge transfer and subsequent coupling reaction (Figure 4C). Their reactivity in the onephoton-mode (366 nm) reactions is achieved through the large population of excited maleimide. In two-photon mode, the maleimide excitation is rare, and the decay to the ground state is more likely than the diffusion-driven reaction of [2+2] cycloaddition. Our experiments support this statement because no hydrogel formation was observed, even at high laser intensities (>500 mW at 720 nm, with peak power density is 62.5 kW/12 × 10−10 cm2 and the energy of the pulse is 5.2 J/ cm2). The [2+2] cycloaddition can also proceed through the donor− acceptor complex if such a complex is present in the reaction mixture.27 The diffusion step is neglected in the DAC reaction pathway, and the light-induced charge separation should lead to a fast reaction because of the molecules’ close proximity (Figure 4C). This route does exist for maleimides, as demonstrated by their reactivity in [2+2] cycloaddition under 405 nm excitation (Figure S16−S17, S30), but does not substantially contribute to the maleimide reactivity in one-photon mode because of a low

Figure 4. Formation of donor−acceptor complex. (A) Schematic of molecular orbital diagram for a donor−acceptor complex; λCT is charge transfer transition wavelength; (B) proposed mechanism of maleimide coupling through symmetry breaking disproportionate to donor and acceptor; (C) electron transfer mechanism of [2+2] cycloaddition reaction of maleimides through diffusing and donor−acceptor pathways.

observed as the appearance of a characteristic charge transfer (CT) absorbance band in the higher-wavelength region of the UV−vis spectrum. The appearance of the CT absorbance is an important characteristic of DAC formation, which differentiates it from other forms of molecular dimers (Figure 4A). Maleimide compounds have been suspected for the formation of DAC in homo-14 and heteromolecular20 systems; however, the CT absorbance was not considered significant. The aggregation of the maleimides could be more complex than a simple dimer DAC because the observed nonlinearity in the plot of absorbance at 400 nm vs the sample concentration (Figure 3C) could be associated with higher-order aggregate formation, analogous to the case of porphyrins.21 Although the CT transition band in the UV−vis spectra is ambiguous because of its low absorbance, the characteristic fluorescence of such transitions (Figure 2B) indicates presence of a DAC in both PEG-Mal4 and its synthetic precursor. DAC formation has been described only for heterogeneous systems, whereas our system is homogeneous; that is, the maleimide groups act as donor and acceptor. Our observation of a DAC implies a symmetry-breaking process for complex formation, which is formally forbidden and, to the best of our knowledge, has not been described for a DAC. Nevertheless, symmetry-breaking processes in photochemistry are well10186

DOI: 10.1021/jacs.7b04484 J. Am. Chem. Soc. 2017, 139, 10184−10187

Journal of the American Chemical Society

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DAC concentration and low absorbance. In two-photon mode, the DAC pathway is favored because even the rare excitation event in DAC would lead to the coupling reaction, resulting in a steady product appearance; that is, close molecule proximity is a major factor in two-photon reactivity. Although the concentration of the maleimide DAC in two-photon mode is the same, the cross section (two-photon absorbance) must be several orders of magnitude higher compared to that of single maleimide molecules.31,32 As shown, π-conjugated donor−acceptor pairs have cross sections more than 2 orders of magnitude larger than the cross sections the donor and acceptor in the absence of conjugation.32 Because the DAC formation/dissociation and the charge separation (ionic pair formation) are subpicosecond processes,33 millisecond of irradiation (100 fs pulse laser) of the sample is adequate for sufficient product formation and its corresponding visualization, which is observed in our experiments (Figure 1D and S3−S7). The enhanced probability of the DAC excitation event in two-photon mode and absence of the diffusion-controlled step promote the [2+2] cycloaddition reaction pathway through the DAC. Our results reveal a mechanism of two-photon [2+2] cycloaddition reactions of maleimides where the reactivity is dictated by excitation of a transition with a low absorbance, formerly ignored in classical photochemistry. We hypothesize and provide evidence this transition should be assigned to the formation of an undescribed symmetry-breaking DAC, an alternative pathway in [2+2] cycloaddition reactions. This DAC is stabilized by aqueous solutions (solvation energy), which explains poor reactivity of the “model compound” in DMF compared to PEG-Mal4 in water. Though the photochemistry of such photoreactions requires further investigation, the twophoton polymerization of the PEG-Mal4 was surprisingly efficient in physiological fluids at millimolar concentration.

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REFERENCES

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b04484.

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AUTHOR INFORMATION

Corresponding Author

*[email protected] ORCID

Mikhail V. Tsurkan: 0000-0003-4000-3890 Michael Schlierf: 0000-0002-6209-2364 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.J. and M.V.T. contributed equally. The authors thank Prof. Dr. Alexander Tarnovsky from the Center for Photochemical Science at Bowling Green University, USA, Dr. Igor L. Zheldakov from Spectra Group Limited, USA, and Prof. Dr. Yixin Zhang from Center for Molecular Bioengineering at TU, Dresden, Germany for their valuable discussions of photochemistry and for reviewing the paper. Financial support by the Bundesministerium für Bildung und Forschung (BMBF) through grants 03Z2EN11 and 03Z2EN512 both to M.S. is gratefully acknowledged. 10187

DOI: 10.1021/jacs.7b04484 J. Am. Chem. Soc. 2017, 139, 10184−10187