Peptidic "Molecular Beacon" for Collagen.

Collagen-mimetic peptides (CMP) have been invaluable tools for understanding the structure and function of collagen, which is the most abundant protein in animals. CMPs have also been developed as probes that detect damaged collagen because of the specificity required to form a collagen triple helix. These probes are not, however, ratiometric. Here, we used EPR spectroscopy to determine the end-to-end distances of CMPs that do not form stable homotrimeric helices. We found that those distances are shorter than the distances in the context of a collagen triple helix, suggesting their potential utility as a "molecular beacon" and guiding the choice and location of a pendant fluorophore-quencher pair. We then showed that a molecular beacon based on a glycine-(2S,4S)-4-fluoroproline-(2S,4R)-4-hydroxyproline tripeptide repeat and EDANS-DABCYL pair enabled the ratiometric detection of its binding to both other CMPs and natural mammalian collagen. These results provide guidance for the development of a new modality for detecting damaged collagen in physiological settings.

Resonant Mixing Dynamic Nuclear Polarization.

We propose a mechanism for dynamic nuclear polarization that is different from the well-known Overhauser effect, solid effect, cross effect, and thermal mixing processes. We term it Resonant Mixing (RM), and we show that it arises from the evolution of the density matrix for a simple electron-nucleus coupled spin pair subject to weak microwave irradiation, the same interactions as the solid effect. However, the SE is optimal when the microwave field is off-resonance, whereas RM is optimal when the microwave field is on-resonance and involves the mixing of states by the microwave field together with the electron-nuclear coupling. Finally, we argue that this mechanism is responsible for the observed dispersive-shaped DNP field profile for trityl samples near the electron paramagnetic resonance center.

Coherent Dynamic Nuclear Polarization using Chirped Pulses.

This paper presents a study of coherent dynamic nuclear polarization (DNP) using frequency swept pulses at 94 GHz which optimize the polarization transfer efficiency. Accordingly, an enhancement ε ∼ 496 was observed using 10 mM trityl-OX063 as the polarizing agent in a standard 6:3:1 d8-glycerol/D2O/H2O glassing matrix at 70 K. At present, this is the largest DNP enhancement reported at this microwave frequency and temperature. Furthermore, the frequency swept pulses enhance the nuclear magnetic resonance (NMR) signal and reduce the recycle delay, accelerating the NMR signal acquisition.

Amplified Overhauser DNP with Selective Deuteration: Attenuation of Double-Quantum Cross-Relaxation.

We recently used selective 2H labeling of BDPA to investigate the Overhauser Effect (OE) dynamic nuclear polarization (DNP) mechanism in insulating solids doped with 1,3-bis(diphenylene)-2-phenylallyl (BDPA), and established that the α and γ 1H spins on the fluorene rings are responsible for generating a zero quantum (ZQ) mediated positive bulk polarization. Here, we establish that the phenyl 1H spins relax via double-quantum (DQ) processes and therefore contribute negative enhancements which attenuate the OE-DNP. With measurements at different magnetic field strengths, we show that phenyl-d5-BDPA offers >50% improvement in OE-DNP enhancement compared to h21-BDPA attaining a maximum of ∼90 at 14.1 T and 5 kHz MAS, the highest observed OE-DNP enhancement to date under these conditions. The approach may be utilized to optimize other polarizing agents exhibiting an OE, an important DNP mechanism with a favorable field and spinning frequency dependence.

Integrated, Stretched, and Adiabatic Solid Effects.

This paper presents a theory describing the dynamic nuclear polarization (DNP) process associated with an arbitrary frequency swept microwave pulse. The theory is utilized to explain the integrated solid effect (ISE) as well as the newly discovered stretched solid effect (SSE) and adiabatic solid effect (ASE). It is verified with experiments performed at 9.4 GHz (0.34 T) on single crystals of naphthalene doped with pentacene-d14. It is shown that the SSE and ASE can be more efficient than the ISE. Furthermore, the theory predicts that the efficiency of the SSE improves at high magnetic fields, where the EPR line width is small compared to the nuclear Larmor frequency. In addition, we show that the ISE, SSE, and ASE are based on similar physical principles and we suggest definitions to distinguish among them.

Overhauser Dynamic Nuclear Polarization with Selectively Deuterated BDPA Radicals.

The Overhauser effect (OE), commonly observed in NMR spectra of liquids and conducting solids, was recently discovered in insulating solids doped with the radical 1,3-bisdiphenylene-2-phenylallyl (BDPA). However, the mechanism of polarization transfer in OE-DNP in insulators is yet to be established, but hyperfine coupling of the radical to protons in BDPA has been proposed. In this paper we present a study that addresses the role of hyperfine couplings via the EPR and DNP measurements on some selectively deuterated BDPA radicals synthesized for this purpose. Newly developed synthetic routes enable selective deuteration at orthogonal positions or perdeuteration of the fluorene moieties with 2H incorporation of >93%. The fluorene moieties were subsequently used to synthesize two octadeuterated BDPA radicals, 1,3-[α,γ-d8]-BDPA and 1,3-[ß,δ-d8]-BDPA, and a BDPA radical with perdeuterated fluorene moieties, 1,3-[α,ß,γ,δ-d16]-BDPA. In contrast to the strong positive OE enhancement observed in degassed samples of fully protonated h21-BDPA (ε ∼ +70), perdeuteration of the fluorenes results in a negative enhancement (ε ∼ -13), while selective deuteration of α- and γ-positions (aiso ∼ 5.4 MHz) in BDPA results in a weak negative OE enhancement (ε ∼ -1). Furthermore, deuteration of ß- and δ-positions (aiso ∼ 1.2 MHz) results in a positive OE enhancement (ε ∼ +36), albeit with a reduced magnitude relative to that observed in fully protonated BDPA. Our results clearly show the role of the hyperfine coupled α and γ 1H spins in the BDPA radical in determining the dominance of the zero and double-quantum cross-relaxation pathways and the polarization-transfer mechanism to the bulk matrix.