[Babcock_Hall]

Our yield has been a little above 50% in the last two peptide coupling reactions.  The reactants are a C-protected amino acid and N-Boc-Ala-OSu, obtained commercially.  DIPEA was also present.  I reviewed the TLC results with my student just now, and the first reaction showed a single spot that was not coincident with either of the two reactants.  The TLC results for the second reaction were less straightforward to interpret, but I am inclined to say that more than one spot was present.  The H-1 NMR of the starting amine looked fine, and we are checking the commercial product now.  We have one other commercial succinimide ester that we could try, and we are considering a model reaction.  I am cross-posting this at Chempros.
EDT
When we used to make amides using 4-nitrophenylesters, we would sometimes follow the progress of the reaction spectrophotometrically. IIRC we pulled the 4-nitrophenolate ion (maximum wavelength of absorption about 400 nm; molar absorptivity about 18,400) into an aqueous buffer to quantitate it. In principle spectrophotometry could also be used to follow the reaction of an NHS ester, after extracting N-hydroxysuccinimide into aqueous solution, although NaOH should be avoided (Miron T, Wilchek M 1982 Analytical Biochemistry 126:433-435). The molar absorptivity of the anion is reported to be 8600-9700, and the pK_a of N-hydroxysuccinimide is reported to be 6.0. The anion absorbs maximally around 260 nm, where many other substances also absorb. It might be more trouble than it is worth to use spectrophotometry. HILIC has been used. DOI: 10.1039/c5ay00042d

[Babcock_Hall]

"...after extracting N-hydroxysuccinimide into aqueous solution..."  It might be possible simply to dilute into aqueous buffer.

[Babcock_Hall]

I did some scratch calculations, and one microliter of the reaction would produce sufficient absorbance.  I might work at pH 7 instead of 8, and correct the molar absorptivity downward slightly for incomplete ionization.  BTW, I suspect that this method would not work as well when using NHS esters to modify proteins because the protein has UV absorbance.

[Babcock_Hall]

https://onlinelibrary.wiley.com/doi/full/10.1002/bip.20013
I am considering using 10% DMAP as a catalyst, which sounds reasonable (the reference above concerns polydepsipeptides; I have not yet done a more thorough literature search).  I am happy to share my scratch calculations regarding UV absorption if anyone is interested.

[Babcock_Hall]

I set up two model reactions yesterday.  Both had 2 equivalents of Boc-Ala-OSu  and one equivalent of Boc-Orn-OH.  One had 10% DMAP.  The Boc-Ornithine was not entirely soluble in THF at 0.15 M, probably owing to the presence of the carboxylic acid.   I followed the reaction using TLC (85:15 EtOAc/methanol) with detection using either phosphomolydate or ninhydrin.  I also followed by taking 1 µL of reaction into 999 µL of pH 7 buffer and scanning and recording the absorbance at 260 nm.  I started the reactions late in the afternoon yesterday and ran overnight.

The ornithine derivative stayed at the baseline and was detectable with ninhydrin and PMA.  The alanine active ester was also ninhydrin-positive, which I tentatively attribute to loss of the BOC group upon heating, which would produce a free amine capable of reacting with ninhydrin.  By ninhydrin each reaction produced a spot that was slower than Boc-Ala-OSu, but ahead of each spot there was a weaker spot with a slightly lower R_f than the starting material.  By PMA similar spots showed up for the standards and for the products.  There was an additional, relatively slow spot that showed up in each product lane that might be N-hydroxysuccinimide (I don't have a standard).  The only surprising result is that I expected to see a more obvious spot for Boc-Ala-OSu in the two product lanes, given that it was in excess.  I did see an extra spot in each product lane, and perhaps there is enough error in the R_f values to allow the conclusion that this is leftover starting material.

The initial time points in the A₂₆₀ experiment both showed strong absorbance, which was unexpected (there is probably a contaminant, possibly NHS itself).  Absorbance increased at nearly the same rate, based on time points taken roughly 5 hours into the reaction and overnight.  I plan to take one more time point each, to see whether or not the absorbance increases further.  Although I have not seen difference between (+)DMAP and (-)DMAP, this is just one experiment.  I chose pH 7 so that most of the N-hydroxysuccinimide would be in its conjugate base form.

I like the idea of increasing the number of equivalents of Boc-Ala-OSu from 1.0 to 1.5-2.0, provided that the remainder can be separated chromatographically.  I think that the use of absorbance over time to look for completeness has promise in some circumstances (assuming that one has access to a spectrophotometer), but it needs a little experimental tweaking.  If I had it to over again, I would probably have used benzylamine as my amine, but I would have had to adjust the absorbance experiment for the background absorption due to benzylamine.
EDT
Assuming that all of the initial (time zero) absorbance that I saw was from contaminating N-hydroxysuccinimide (NHS), its concentration was about 62 mM.  I have not worked out the mole fraction yet, but I would guess it to be about 20-25%, relative to Boc-Ala-OSu.

[rolnor]

Nice work!
I think you are getting tired if hearing this, but, you would be much more in touch with you reactions if you could run LC-MS, its a perfect, job for that. TLC is obviously dufficult with free carboxylic acids etc.