Nuclear singlet multimers (NUSIMERs) with long-lived singlet states

We are introducing nuclear spin singlet multimers which are molecules that contain several nuclear singlet states that can be populated at the same time.

50 mM pyrazine. The amount of 2 and 4 in the solutions was calculated to be 100 µM assuming complete derivatization. Thus, the assumed molarity of tripeptide residues would be 12.8 mM. The actual molarity of tripeptide residues has been calculated by comparing the integral of the pyrazine signal with the integral of the methyl Protons of the Alanine. The actual derivatization is 0.69 for 2 and of 0.68 for 4. This results in a mean coverage of about 90 tripeptides per NUSIMER.

On the accessibility of the singlet state in (2)
As mentioned in the main article the singlet state in the signal at 3.87 ppm was not accessible any more upon PBS addition. As shown in figure S1, measurements performed on a BRUKER 400 MHz machine in D 2 O showed a difference in chemical shift in the signal at 3.80 ppm which was not visible in PBS solution anymore. Figure S1:Expansion of the 1 H NMR spectrum of 2 showing from left to right the signal of the CH proton of alanine (quartet centered at 3.97ppm) and of the CH 2 protons of the two glycine residue at 3.87 ppm and 3.81 ppm, respectively . In the spectrum measured in D 2 O (blue) a chemical shift difference is visible, whereas in PBS solution (red) the chemical shift difference can not be resolved anymore.
Overall, this region of the NMR spectrum seems to be highly influenced by the presence of PBS. In the same spectrum the signals arising from the methyl group of the alanine, as well as the signals of the Boc protection group, show a change in the other direction, showing higher resolution and the appearance of a splitting of the signals (figure S2). This observation leads us to assume a strong interaction of the NUSIMERs with the ions in the PBS solution. Further investigation of this phenomenon will be subject of our future research. Here, we speculate that in presence of PBS, the interaction leading to a chemical shift difference in the glycine signal at 3.81 ppm is influencing the signal at 3.87 ppm in the same way, making the singlet state inaccessible.
Singlet life-times have been investigated in D 2 O for the signal at 3.81 ppm and a singlet relaxation time of T s =2.51 ± 0.04 s. Removing the spin lock caused T S to decrease significantly to T s =0.73 ± 0.02 s. This reflects the observed change in chemical shift difference. For comparison T S have been determined in H 2 O (5 % D 2 O) solutions without the addition of PBS for 2 as well. The singlet state in both signals has been accessible with T s =1.08 s and T s =3.46 s in the signals at 3.95 ppm and 4.02 ppm respectively, showing that the observed change in accessibility can be attributed to the PBS and is not dependent on the deuteration level of the solvent.

Estimation of the recovered magnetization
The magnetization recovered after the end of the experiments have been estimated. For each of the signals the SLIC 1 pulse lengths and powers have been optimized and the singlet evolution time was set to 200 ms. For the protons of the glycine further away from the alanine residue we found that 5% of magnetization could be recovered (SLIC pulse length 310 ms, SLIC pulse power 6.6e -6 W). For the glycine protons closer to the alanine residue, 41% of the initial magnetization could be recovered (SLIC pulse length 50 ms, SLIC pulse power 8e -6 W). The theoretical maximum with the SLIC technique is 66% percent and our experiments show that optimization of the chemical shift difference between the two protons of interest can enhance the obtainable signal in the future.

Measurements of T 1
For the measurement of the T 1 relaxation times, an inversion recovery experiment has been conducted. The obtained plots are listed below. The values given in the main article are the mean values taken from the values obtained from two different samples.                                             NMR spectra of (2) and (4) Figure S48: Proton spectrum of a 100 µM solution of 2 in D 2 O with presaturation on the solvent signal at a magnetic field of 7.05 T. Two scans were run in this experiment with an inter-scan delay of d1=5 s. The 90° pulse was p1=16 µs.    IR spectra of the starting material as well as of (2) and (4) Figure S54: IR spectrum of the starting material G5-PAMAM-NH 2 .