PS-MBHA resin beads (75- to 150-m m diameter) were purchased from Aldrich (2% cross-linking, loading 1.1 mmol/g) and Polymer Labs (1% cross-linking, loading 1.6 mmol/g). Methylene chloride was distilled over P₂O₅ when used for allylic alkylations, otherwise solvents were used as received. The EBES linker was synthesized similarly to the reported procedure with the substitution of Boc₂O for Fmoc succinimide (1). The Boc-protected EBES was obtained as a clear oil. The diazaphospholane 1 was synthesized and resolved as described previously (2). 1,3-Dimethylallyl acetate was synthesized by an established route (3). Parallel solid-phase reactions were performed in PolyPrep chromatography columns purchased from Bio-Rad. Tetrabutylammonium fluoride was purchased from Fisher. Amino acids were purchased from Novabiochem and all other chemicals were purchased from Aldrich. General procedures for coupling reaction were taken from the Novabiochem catalog (4).

Magic-angle spinning NMR was performed with a Varian Nanoprobe on an INOVA 500. Routine NMR experiments were carried out on a Bruker AC-300. NMR data are reported as follows: chemical shift (multiplicity [b = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet], integration, and peak identity, if known).

Solution-Phase Synthesis (General Procedure). The dipeptide was synthesized by the standard DIC/HOBt method using Boc protection. Boc-protected alanine was coupled with the methyl ester of phenylalanine with 1 eq each of hydroxybenzotriazole (HOBt) and DIC (1,3-diisopropylcarbodiimide) in 50 ml of CH₂Cl₂. The solution was stirred under N₂ overnight. The organic layer was rinsed with 75 ml of saturated NaHCO₃, twice with 75 ml of 1 M HCl, once with 75 ml of saturated NaHCO₃, and once with 75 ml of water. The organic layer was dried over MgSO₄ and the solvent was removed under vacuum. The Boc-protected product was purified by flash chromatography (2% methanol in CH₂Cl₂). Boc was removed by mixing the peptide with 50% trifluoroacetic acid (TFA) in CH₂Cl₂ for 30 min. The deprotection of the peptide Ala-Met-OMe used the scavengers 2.5% triisopropylsilane and 2.5% water (5). The dipeptide Ala-Met-OMe was purified by precipitation with cold diethyl ether and dried under vacuum. The rest of the dipeptides were purified by removal of solvent under vacuum.

The carboxylic acid diazaphospholane 1 (0.41 mmol) was placed in a Schlenk line with 3 eq of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). Fifty milliliters of CH₂Cl₂ was added to the Schlenk line. A solution of 3 eq of the dipeptide with 8 eq of DIEA in 10 ml of CH₂Cl₂ was added to the stirring mixture. The resulting yellow solution was briefly placed under vacuum and then the space above the solution was filled with nitrogen. The solution was stirred overnight. The solvent was then removed under vacuum and the residue was dissolved in 100 ml ethyl acetate. The organic layer was rinsed once with a saturated solution of NaHCO₃, twice with a 2 M HCl solution, once with saturated NaHCO₃, and once with water. The organic layer was dried over MgSO₄ and the solvent was removed to leave a yellow oil.

(R,R)-Val-Ala-OMe. The product was purified by flash chromatography with 1:1 ethyl acetate/methylene chloride as eluent. Yield = 85% of a white solid. ¹H NMR (CDCl₃): d 0.96 (d, 6H, J = 6.9 Hz, NCHCH₃), 0.96 (d, 3H, J = 6.9 Hz, CH(CH₃)₂), 1.00 (d, 3H, J = 6.9 Hz, CH(CH₃)₂), 1.16 (d, 3H, J = 7.4 Hz, CH(CH₃)₂), 1.37 (d, 3H, J = 7.1 Hz, CH(CH₃)₂), 2.1 (b, 2H, CH(CH₃)₂), 3.67 (s, 3H, CO₂CH₃), 3.71 (s, 3H, CO₂CH₃), 4.4 (m, 4H, NHCH), 6.07 (d, 1H, J = 8.1 Hz), 6.60 (d, 1H, J_H-P = 18.4 Hz, PCHN), 6.66 (t, 1H, J = 7.9 Hz), 6.8-7.5 (m, 15H), 7.64 (m, 1H), 7.83 (m, 2H), 8.22 (m, 1H), 8.40 (m, 1H); ¹³C NMR (CDCl₃): d 17.8 (s, CH₃), 18.0 (s, CH₃), 18.7 (s, 2C, CH₃), 19.4 (s, CH₃), 19.5 (s, CH₃), 30.9 (s, CH(CH₃)₂), 31.2 (s, CH(CH₃)₂, 48.2 (s, CHCH₃), 48.7 (s, CHCH₃), 52.5 (s, CO₂CH₃), 52.7 (s, CO₂CH₃), 59.0 (d, J_C-P = 21 Hz, PCHN), 59.5 (s, 2C, NHCHCH), 62.2 (d, J_C-P = 34 Hz, PCHN), peaks at 124-140 ppm have not been assigned due to the complexity, 156.3 (s, CONN), 156.9 (s, CONN), 169.0 (s, CONH), 169.3 (s, CONH), 170.7 (s, CONH), 171.0 (s, CONH), 173.1 (s, CO₂CH₃), 173.4 (s, CO₂CH₃); ³¹P NMR (CDCl₃): d 1.9 ppm. Exact mass measurement (EMM) calcd for C₄₈H₅₃N₆O₁₀PNa [M + Na]⁺: 927.3459, found: 927.3414.

(R,R)-Ala-Ala-OMe. The crude product was purified by flash chromatography with ethyl acetate as eluent. Yield = 60% of a white solid. ¹H NMR (CDCl₃): d 1.33 (d, 3H, J = 7.1 Hz, CHCH₃), 1.38 (d, 3H, J = 7.1 Hz, CHCH₃), 1.45 (d, 6H, J = 7.0 Hz, CHCH₃), 3.71 (s, 3H, CO₂CH₃), 3.75 (s, 3H, CO₂CH₃), 4.4-4.7 (m, 4H, NCHCH₃), 6.17 (d, 1H, J = 7.8 Hz), 6.72 (m, 2H), 6.80 (d, 1H, J_H-P = 18.5 Hz, PCHN), 6.85-7.4 (m, 14H), 7.62 (d, 1H, J = 6.5 Hz), 7.76 (broad, 1H, NHCH), 7.84 (m, 2H), 8.24 (m, 1H), 8.38 (m, 1H); ¹³C {¹H} NMR (CDCl₃): d 18.1 (s, CHCH₃), 18.2 (s, CHCH₃), 18.3 (s, CHCH₃), 18.4 (s, CHCH₃), 48.3 (s, CHCH₃), 48.4 (s, CHCH₃), 49.4 (s, 2C, CHCH₃), 52.6 (s, CO₂CH₃), 52.7 (s, CO₂CH₃), 59.3 (d, J_C-P = 15 Hz, PCHN), 62.2 (d, J_C-P = 37 Hz, PCHN), peaks at 125-140 ppm have not been assigned due to the complexity, 156.4 (s, CONN), 157.0 (s, CONN), 168.4 (s, CONH), 168.7 (s, CONH), 171.7 (s, CONH), 171.7 (s, CONH), 173.3 (s, 2C, CO₂CH₃); ³¹P NMR (CDCl₃): d 1.6 ppm. EMM calcd for C₄₆H₄₅N₆O₁₀PNa [M + Na]⁺: 871.2833, found: 871.2875.

(R,R)-Ala-Met-OMe. The crude product was purified by flash chromatography with ethyl acetate as eluent. Yield = 10% of a white solid. ¹H NMR (CDCl₃): d 1.48 (d, 6H, J = 6.7 Hz, CHCH₃), 1.9-2.3 (m, 4H, CHCH₂CH₂), 2.05 (s, 6H, SCH₃), 2.49 (m, 4H, CH₂S), 3.73 (s, 3H, CO₂CH₃), 3.76 (s, 3H, CO₂CH₃), 4.5-4.8 (m, 4H, NHCH), 6.19 (d, 1H, J = 7.8 Hz), 6.6-7.3 (m, 13H), 7.37 (m, 3H), 7.63 (d, 1H, J = 7.0 Hz), 7.84 (m, 2H), 8.0 (b, 1H, NHCH), 8.24 (m, 1H), 8.38 (m, 1H); ¹³C{¹H} NMR (DMSO-d₆): d 14.6 (s, CHCH₂CH₂), 14.7 (s, CHCH₂CH₂), 17.6 (s, CHCH₃), 17.7 (s, CHCH₃), 29.4 (s, CH₂CH₂S), 29.5 (s, CH₂CH₂S), 30.5 (s, 2C, SCH₃), 48.3 (s, CHCH₃), 48.5 (s, CHCH₃), 50.8 (s, CHCH₂), 50.9 (s, CHCH₂), 51.8 (s, CO₂CH₃), 51.9 (s, CO₂CH₃), 58.0 (d, J_C-P = 21 Hz, PCHN), 61.3 (d, J_C-P = 36 Hz, PCHN), peaks at 125-140 ppm have not been assigned because of the complexity, 155.0 (s, CONN), 155.6 (s, CONN), 167.4 (s, CONH), 167.7 (s, CONH), 172.0 (s), 172.1 (s), 172.3 (s), 172.5 (s); ³¹P NMR (DMSO-d₆): d 3.0 ppm. EMM calcd for C₄₈H₅₃N₆O₁₀PS₂Na [M + Na]⁺: 991.2900, found: 991.2875.

Solid-Phase Synthesis of (R,R)-Ala. The MBHA resin (250 mg, 1.6 mmol/g) was loaded onto a glass frit that was attached to a nitrogen/vacuum line. The resin was combined with 8 ml of DMF for 1 h while agitating with nitrogen bubbling through the frit (referred to as mixing for the rest of this procedure). DIEA (1 ml) was added to the solution and the solution was mixed for 45 min. The solution was drained by applying a vacuum through the frit. The resin was rinsed three times with DMF (10 ml, 30 min). The standard rinsing protocol for the synthesis was to agitate with nitrogen for 30 min and then drain. The Boc-protected linker (0.50 g, 1.4 mmol) was combined with HOBt (0.20 g, 1.5 mmol) in 8 ml of DMF. The solution was added to the resin and mixed for 5 min. DIC (0.22 ml, 1.4 mmol) was added to the mixture and the reactants were mixed overnight.

The mixture was drained and the resin was rinsed twice with DMF and MeOH, and once with CH₂Cl₂. The resin was rinsed once more with DMF. The Boc protecting group was removed by treating the resin with 30% TFA in CH₂Cl₂ with 2% anisole for 30 min. The mixture was drained and rinsed twice each with CH₂Cl₂, DMF, and MeOH. The solution was rinsed once more with DMF. Fmoc-alanine (0.50 g, 1.6 mmol) and HOBt (0.22 g, 1.6 mmol) were combined in 8 ml of DMF. This solution was added to the resin and mixed. After 5 min DIC (0.25 ml, 1.6 mmol) was added and the reactants were mixed overnight. The solution was drained and the resin was rinsed twice with DMF and MeOH. The Fmoc protecting group was removed by adding a solution of 20% piperidine in DMF and mixing for 30 min. The solution was drained, the resin was rinsed with DMF, and the deprotection was repeated. After draining the solution, the resin was rinsed twice with DMF, twice with MeOH, and once with CH₂Cl₂. The resin was rinsed once more with DMF.

The (R,R)-carboxylic acid diazaphospholane 1 (0.86 g, 1.6 mmol) was combined with HOBt (0.22 g, 1.6 mmol) in 10 ml of DMF. The resulting solution was added to the resin, followed by DIC (0.25 ml, 1.6 mmol). The reactants were mixed overnight. The next day the solution was drained and rinsed twice with DMF, once with MeOH, and once again with DMF. Alanine methyl ester (0.28 g, 2.0 mmol) was combined in a beaker with DIEA (0.35 ml, 2.0 mmol) in 8 ml of DMF. After mixing the solution, HOBt (0.27 g, 2.0 mmol) was added. This solution was added to the resin, followed by DIC. The reactants were mixed overnight and the solution was drained. Rinsing the resin twice each with DMF, MeOH, and CH₂Cl₂ provided the product. The product was collected from the glass frit by suspending it in CH₂Cl₂ and transferring the resin to a vial. The resin was dried under vacuum overnight. The product was characterized by ³¹P MAS-NMR (DMSO-d₆): d 1.4, 1.8 ppm.

Parallel Synthesis of Immobilized Diazaphospholanes. The MBHA resin (75 mg) was allowed to swell in 8 ml of DMF for 1 h. The resin was neutralized by treatment with 1 ml of DIEA in 20 ml of DMF for 2 h followed by rinses. Five equivalents of the Boc-protected EBES linker was added to the resin by dissolving it in 2 ml of DMF with HOBt (5 eq) followed by addition to the resin. After 2 min, DIC (5 eq) was added to the mixture and the reaction was allowed to proceed overnight. After rinsing with DCM, DMF, and MeOH, the Boc-protected linker was deprotected by adding 30% TFA in DMF with 2% anisole and mixing for 1 h. The resin was rinsed according to the general protocol. The resins were then coupled with Fmoc-protected AA². Five equivalents of the Fmoc-amino acids was dissolved in 2 ml of DMF with HOBt (5 eq). The DIC (5 eq) was added and the resulting solution was added to the resin. The samples were capped and mixed on a shaker overnight.

The next day the mixtures were drained and rinsed twice with CH₂Cl₂, once with DMF, twice with MeOH, and once with DMF. The resins were treated with a 20% piperidine solution in DMF and the solution was allowed to react for 30 min. The resin was rinsed with DMF and the deprotection was repeated. The resins were rinsed twice with DMF, twice with MeOH, and twice with CH₂Cl₂. The resins were coupled with Fmoc-protected AA¹ following the same procedure as detailed above. The next day the resins were isolated and deprotected by using the same procedure.

The resin samples were placed in a glove bag and purged with nitrogen for thirty min. All subsequent reactions were set up in a glove bag under nitrogen. The (R,R)-carboxylic acid diazaphospholane (5.3 g, 9.9 mmol) and HOBt (1.3 g, 9.6 mmol) were combined and purged with nitrogen. A 4:1 mixture of CH₂Cl₂ and DMF was added to the solids and the solution was mixed for five min. DIC (1.5 ml, 9.6 mmol) was added to this solution and thoroughly mixed under nitrogen. A 2.5 ml portion of the solution was added to each of the 20 resin samples. The solutions were capped and placed on a shaker overnight.

The solutions were drained and rinsed once with CH₂Cl₂. The AA₁ t-butyl esters (5 eq) and HOBt (5 eq) were combined and then a mixture of 4:1 CH₂Cl₂ and DMF was added and followed by 5 equivalents of DIEA. The samples that had no AA₂ were treated with AA₁ methyl esters instead of the t-butyl esters. The solutions were thoroughly mixed and DIC (5 eq) was added to each. The solutions were then added to their respective resin samples. The columns were capped, removed from the glovebag, and mixed on a shaker overnight.

The solutions were drained and rinsed once with CH₂Cl₂, twice with DMF and twice with CH₂Cl₂. A solution of 95% TFA/2.5% triisopropylsilane/ 2.5% water was added to each sample in the glove bag to remove the t-butyl ester protecting group. The mixture was placed on a shaker and the reaction was allowed to proceed for 3 h. The solutions were drained and the resins were rinsed twice each with CH₂Cl₂, DMF, MeOH and again with CH₂Cl₂. The AA₂ methyl esters were coupled using the same procedure described above. After mixing overnight, the samples were rinsed twice with DMF, twice with MeOH and three times with CH₂Cl₂. The samples were dried under vacuum overnight and stored under nitrogen.

1. Song, A., Zhang, J., Lebrilla, C. B., & Lam, K. S. (2003) J. Am. Chem. Soc. 125, 6180-6188.

2. Clark, T. P. & Landis, C. R. (2003) J. Am. Chem. Soc. 125, 11792-11793.

3. von Matt, P., Loiseleur, O., Koch, G., Pfaltz, A., Lefeber, C., Feucht, T. & Helmchen, G. (1994) Tetrahedron Asymmetry, 5, 573-584.

5. White, P., Dörner, B. & Steinauer, R., eds. (2002) in Novabiochem 2002-2003 Catalog (Novabiochem, Darmstadt, Germany), pp. 3.1-3.38.

5. Mehta, A., Jaouhari, R., Benson, T. J. & Douglas, K. T. (1992) Tetrahedron Lett. 33, 5441-5444.