CDAP (1-cyano-4-dimethylaminopyridine tetrafluoroborate) is employed in the synthesis of conjugate vaccines as a cyanylating reagent. In the published method, which used pH 9 activation at 20 °C (Vaccine, 14:190, 1996), the rapid reaction made the process difficult to control. Here, we describe optimizing CDAP activation using dextran as a model polysaccharide. CDAP stability and reactivity were determined as a function of time, pH and temperature. While the rate of dextran activation was slower at lower pH and temperature, it was balanced by the increased stability of CDAP, which left more reagent available for reaction. Whereas maximal activation took less than 2.5 min at pH 9 and 20 °C, it took 10–15 min at 0 °C. At pH 7 and 0 °C, the optimal time increased to >3 h to achieve a high level of activation. Many buffers interfered with CDAP activation, but DMAP could be used to preadjust the pH of polysaccharide solutions so that the pH only needed to be maintained. We found that the stability of the activated dextran was relatively independent of pH over the range of pH 1–9, with the level of activation decreased by 40–60% over 2 h. The use of low temperature and a less basic pH, with an optimum reaction time, requires less CDAP, improving activation levels while making the process more reliable and easier to scale up.

This paper presents the observation of four-top-quark ($$t\bar{t}t\bar{t}$$ t t ¯ t t ¯ ) production in proton-proton collisions at the LHC. The analysis is performed using an integrated luminosity of 140 $$\hbox {fb}^{-1}$$ fb - 1 at a centre-of-mass energy of 13 TeV collected using the ATLAS detector. Events containing two leptons with the same electric charge or at least three leptons (electrons or muons) are selected. Event kinematics are used to separate signal from background through a multivariate discriminant, and dedicated control regions are used to constrain the dominant backgrounds. The observed (expected) significance of the measured $$t\bar{t}t\bar{t}$$ t t ¯ t t ¯ signal with respect to the standard model (SM) background-only hypothesis is 6.1 (4.3) standard deviations. The $$t\bar{t}t\bar{t}$$ t t ¯ t t ¯ production cross section is measured to be $$22.5^{+6.6}_{-5.5}$$ 22 . 5 - 5.5 + 6.6  fb, consistent with the SM prediction of $$12.0 \pm 2.4$$ 12.0 ± 2.4 fb within 1.8 standard deviations. Data are also used to set limits on the three-top-quark production cross section, being an irreducible background not measured previously, and to constrain the top-Higgs Yukawa coupling and effective field theory operator coefficients that affect $$t\bar{t}t\bar{t}$$ t t ¯ t t ¯ production.

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