500 mhz 1h nmr spectrometer

Proton nuclear magnetic resonance (¹H-NMR) spectroscopy was employed to characterize the Gem-SPDP conjugate in order to confirm the conjugation, as well as, the purity of the synthesized material. A sample (3 mg) of column-purified Gem-SPDP conjugate was dissolved in deuterated dimethylsulfoxide (DMSO) and was characterized using a 500 MHz ¹H-NMR spectrometer (Varian Inc., CA). For the liquid chromatography/mass spectrometry (LC/MS) analysis, a 1 mg/ml solution of the conjugate in methanol was further diluted 1000× with methanol and characterized on Agilent 1200 Infinity series LC coupled with 6120 quadrupole MS (Agilent Technologies, Santa Clara, CA, USA) operating in positive ion mode to obtain the mass spectra of the conjugate.

The biotinylated poly(ethylene) glycol (PEG) polymer was prepared by
dissolving 1g of α-hydroxy-x-amine PEG 2000
(JenKem) in 50mM sodium biocarbonate (pH=8.2.) An equimolar amount
(0.25 g) of NHS–biotin (Sigma) was then added dropwise and reacted
for 2 hours at room temperature. Unreacted NHS-biotin was dialyzed away
using 1,000 m.w.c.o. tubing and purified product was lyophilized.
Biotinylation was confirmed using ¹H NMR in deuterated chloroform
with tetramethylsilane (Sigma) used as the internal reference on a Varian
500MHz ¹H NMR spectrometer (Supplementary Figure S1a, b).
Since the biotin was a terminal modification of the polymer, the biotin
makes up 12% of the compound by mass so the splitting patterns of
the biotin peaks were hard to visualize but the relevant peaks are assigned
here ¹H NMR (CDCl₃) δ 4.5–4.4 (m, 1H),
4.3 (m, 1H), 3.2–3.1 (m, 1H), 3.0–2.9 (m, 1H),
2.8–2.7 (m, 1H), 2.3–2.1 (m, 2H). The remaining 6 H appear
from 1.5 – 1.0 but are confounded by residual grease and
solvent.