3.5.4. Coupling of Fmoc-D-*allo*-Ile

Coupling of Fmoc-D-*allo*-Ile was achieved by dissolving Fmoc-D-*allo*-Ile (0.32 mmol, 3.2 eq.), in 0.4 M HATU/DMF (0.75 mL, 0.3 mmol, 3.0 eq.), followed by the addition of DIPEA (105 μL, 0.6 mmol, 6.0 eq.). The coupling cycle consisted of Fmoc deprotection with 20% of piperidine in DMF (twice, 5 and 10 min), a 5 min DMF flow-wash, followed by coupling with preactivated Fmoc-D-*allo*-Ile (3.2 eq.) over 2 × 30 min.

#### 3.5.5. Derivatization with L-FDAA

Fmoc deprotection was achieved by the addition of 20% of piperidine in DMF (twice, 5 and 10 min), a 5 min DMF flow-wash, followed by coupling with L-FDAA reagent, 1% solution in acetone (3.2 eq.), in the presence of DIPEA (105 μL, 0.6 mmol, 6.0 eq.) for 1 h. The resin was washed with acetone (5 × 1 min), dry CH2Cl2 (5 × 1 min), 1:1 CH2Cl2/MeOH (5 × 1 min) and MeOH (2 × 1 min), then dried (vacuum desiccator).

#### 3.5.6. Cleavage of L-FDAA Derivatized Dipeptide from Resin

After swelling the 2-CTC resin for 20 min in dry CH2Cl2 (2 mL) the resin was mixed with 20% hexafluoro-2-propanol (HFIP)/CH2Cl2 (2 mL × 3 × 20 min) and the combined filtrate evaporated in vacuo to yield analytical samples of L-FDAA-D-*allo*-Ile-L-Ala [HRESI(+)MS *m*/*z* 477.1722 [M + Na]+ (calcd for C18H26N6O8Na 477.1704) and L-FDAA-D*allo*-Ile-D-Ala [HRESI(+)MS *m*/*z* 477.1692 [M + Na]+ (calcd for C18H26N6O8Na 477.1704), both of which were shown to be pure by HPLC-DAD-MS (method as described in general experimental section) (Figures S46 and S47).

#### 3.5.7. Marfey's Method #6 Optimized for L-FDAA-D-*allo*-Ile-Ala Diastereomers

An aliquot of Marfey's derivatized analyte (3 μL) (see method #5) was subjected to UPLC-DAD-MS analysis using the same binary solvent system and detection as described above (method #2), but with an isocratic 0.6 mL/min elution at 37% Phase B in A, and with comparison between natural and synthetic Marfey's derivatized dipeptides (Figure S41).
