Lithium hexamethyldisilazide initiated superfast ring opening polymerization of alpha-amino acid N-carboxyanhydrides

Polypeptides have broad applications and can be prepared via ring-opening polymerization of α-amino acid N-carboxyanhydrides (NCAs). Conventional initiators, such as primary amines, give slow NCA polymerization, which requires multiple days to reach completion and can result in substantial side reactions, especially for very reactive NCAs. Moreover, current NCA polymerizations are very sensitive to moisture and must typically be conducted in a glove box. Here we show that lithium hexamethyldisilazide (LiHMDS) initiates an extremely rapid NCA polymerization process that is completed within minutes or hours and can be conducted in an open vessel. Polypeptides with variable chain length (DP = 20–1294) and narrow molecular weight distribution (Mw/Mn = 1.08–1.28) were readily prepared with this approach. Mechanistic studies support an anionic ring opening polymerization mechanism. This living NCA polymerization method allowed rapid synthesis of polypeptide libraries for high-throughput functional screening.


Synthesis of Nε-tert-butyloxycarbonyl-L-lysine NCA (Boc-L-Lys NCA)
Boc-L-Lys NCA was prepared using a procedure identical to that for Boc-D,L-Lys NCA

Synthesis of L-Leucine NCA (L-Leu-NCA)
L-Leu-NCA 7,8 was prepared as a white crystal in 75% yield, from L-leucine using a similar method described above for BLG NCA. 1

Synthesis of β-tert-butyl-L-aspartate NCA (tBu-L-Asp NCA)
tBu-L-Asp NCA 9 was prepared as a white crystal in 55% yield, from L-aspartic acid β-tertbutyl ester using a similar method described above for Boc-D,L-Lys NCA. 1 Figure 96 and 97 below.

Polymer synthesis and characterization
A general synthesis of poly-BLG is described below and all others polymers were synthesized similarly. The polymer length was controlled via the BLG NCA:initiator ratio.  Figure 24).

Polarimetry measurement
Chirality of the deprotected polymers was measured at λ=589 nm using the Autopol® V automatic polarimeter. Deprotected polymers were prepared in milli-Q at final concentration of 10 mg/mL and measured at pH=8 and at 25 ℃. Specific rotation ([α] T λ ) was calculated using the following equation: Where α is the angle of rotation, l is the length of the polarimeter tube (dm), c is polymer concentration (g/100 mL).

Terminal functionalization for polypeptides prepared from LiHMDS-initiated polymerization
After completion of the polymerization reaction, the poly-Boc-D,L-Lys at sidechain amine protected (NHBoc) stage was characterized by GPC for absolute molecular weight measurement.
GPC result indicated the poly-Boc-D,L-Lys have an average polymer length of 20 subunits (DP=20). 4-tert-butylbenzylamine was added as a nucleophilic reagent to the reaction mixture in THF and the reaction was allowed to stir overnight. The reaction mixture was then poured into petroleum ether to precipitate out the resulting polymer as a white solid and the solid was collected after centrifugation. This dissolution-precipitation process was repeated two more times and the white solid was dried under vacuum to get the C-terminal functionalized polypeptide.
The white solid was collected after centrifugation and then was washed with acetonitrile (1 mL × 3) to make sure all N-succinimidyl 3-maleimidopropionate was removed from the polymer. The

Analysis of subunit distribution within amphiphilic co-polymers
To evaluate the subunit distribution within an amphiphilic co-polymer from LiHMDS Total NCA monomer conversion (x axis) was calculated from the equation below.

Antibacterial activity studies
The fresh cultured bacteria in LB (Luria-Bertani) medium at 37 °C was diluted in MH (Mueller-Hinton) medium to a cell density of 2×10 5 CFU/mL as the working suspension.
Two-fold serial dilution of polypeptides was performed in a 96-well plate using MH medium to provide each well of 50 µL solution with polypeptide concentration ranging from 3.13 to 400 µg/mL. An aliquot of 50 µL above bacteria cell working suspension was added to each well, followed by gentle shaking of the plate for 10 second. The plate was incubated at 37 °C for 9 hours, and then the optical density (OD) of each well was measured at 600 nm using a Molecular Devices SpectraMax M2 precision microplate reader. Wells containing only MH medium was used as the blank and wells containing cells in MH without polymer was used as the positive control on the same plate. Measurements were performed in duplicates, and the experiments were repeated at least twice on different days. The percentage of bacteria cell growth in each well was calculated from the equation below and plotted against polypeptide concentration to give the dose-response curves of antibacterial activity for these polypeptides.
The MIC value is defined as the minimum concentration of a polypeptide to inhibit bacterial growth.

NCA stability in THF vs. DMF.
Most of the NCA polymerizations in literatures continued for 2-3 days using DMF or THF as the solvent. However, many NCAs are quite unstable and the decomposition can bring side reactions. 12,13 We analyzed the stability of widely used γ-benzyl-L-glutamate NCA (BLG NCA) and Nε-tert-butyloxycarbonyl-D,L-lysine NCA (Boc-D,L-Lys NCA) in dry DMF and THF at room temperature without addition of any polymerization initiator. We found that BLG NCA is stable in dry THF for at least 22 hours, but its concentration reduced quickly in DMF from the Another possible reason for observed difference in NCA stability is that dry DMF generally may have more water content than does dry THF. Therefore, we carefully measured the water content of anhydrous DMF and THF using a Karl Fisher titrator. The actual water content was found to  [ ] = ln

Supplementary Equation 6
HPLC was also used to measure remaining NCA within the reaction mixture by calculating the relative ratio of peak area between the NCA and the internal standard.

Supplementary Equation 7
Then the polymerization rate was calculated from plotting the natural logarithm of NCA concentration vs. reaction time using below equation.

Polypeptide backbone racemization evaluation
To mimic polypeptides prepared with variable degree of backbone racemization, we