The azido acid approach to β-peptides: parallel synthesis of a tri-β-peptide library by fluorous tagging

Abstract

A small tri-β-peptide library was prepared starting from three enantio- and diastereopure azido acids. Fluorous tagging followed by two cycles of azide reduction, fluorous solid phase extraction (f-SPE), peptide coupling with the original azido acids and f-SPE provided 27 protected azido peptides. Reduction and HPLC purification provided 25 of the 27 targeted tri-β-peptides in acceptable yields and excellent purities.

1. Introduction

Oligomers and polymers of β-amino acids, called β-peptides, are of increasing interest because of their structures, folding patterns and potential biological activities.¹ Most syntheses of β-peptides are patterned after the traditional approach to making α-peptides.² The key building blocks are N-protected β-amino acids, which are joined by an iterative sequence of N-deprotection and amide coupling (Figure 1).

Figure 1.

The “amino acid” and “azido acid” approaches to β-peptides

Nelson and coworkers recently introduced an approach to β-peptides that starts from β-azido acids rather than β-amino acids and involves an iterative sequence of reduction of the azide to an amine followed by union of the amine with the next β-azido acid by amide bond formation.³ α-Peptides have also been made from α-azido acids.⁴ But the azide approach is not used very often because α-amino acids are much more readily available than α-azido acids and because α-azido acid derivatives activated for peptide coupling reactions are prone to racemization. This is unfortunate because atom economy and small size make the azide group attractive compared to protected amines.

For the β-peptide series, enantiopure β-azido acids are readily available by several routes,⁵ and activated β-azido acids for peptide coupling cannot epimerize at the azide-bearing carbon. Thus, the azide approach merits serious consideration as a general approach to β-peptides.

Only a handful of di-β-peptides and a lone tri-β-peptide have been made by the azide approach to date.³ We set out to extend the scope of the method by making a 27-member library of tri-β-peptides in a 3 × 3 × 3 matrix starting from three enantiopure β-azido acids. To expedite the library preparation, we used a light fluorous tag^6,7 on the C-terminus of the first β-azido acid so that intermediates could be quickly purified by fluorous SPE (f-SPE).⁸ We describe herein the results of this study.

2. Results and Discussion

The syntheses and fluorous tagging of the three β-azido acid building blocks are summarized in Scheme 1. β-Lactones 1a–c are readily made in one step, on multi-gram scale, in >90% ee by the cinchona-alkaloid catalyzed cyclocondensation reactions between acyl halides and aldehydes (AAC reaction).³ Reactions of purified β-lactones 1a,b and with NaN₃ in DMF at 50 °C provided β-azido acids 2a,b in quantitative yield. Due to its volatility, β-lactone 1c was not purified; instead, the crude product of the AAC reaction was directly subjected to azide opening, and then the azide was purified. This provided 2c in 65% yield over the two steps. To complete the precursor synthesis, the three β-azido acids (110–140 mg) were esterified with 1.2 equiv of the fluorous PMB alcohol 3 (^FPMB-OH)⁹ in presence of 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide (EDCI) and N,N-dimethylaminopyridine (DMAP) to obtain the three azido-esters 4a–c. These key precursors were purified by standard flash chromatography.

Scheme 1.

Synthesis of azido acid precursors

The synthesis of the tri-β-peptide library is summarized in Scheme 2 and involves two cycles of azide reduction and amide coupling (4 → 5 and 5 → 6), followed by a final hydrogenation/hydrogenolysis (6 → 7) to reduce the terminal azide and remove the fluorous PMB group.

Scheme 2.

Synthesis of the tri-β-peptide library

The azides were reduced by a Staudinger procedure.^10,11 The three azido-esters 4a–c were treated with 1.2 equiv of triphenylphosphine in THF under microwave irradiation at 120 °C, 250 W. After 5 min, ³¹P NMR spectroscopic analysis showed complete conversion to the azaylide (see Supplementary Material). Water (20 equiv) was added and the mixtures were again irradiated in the microwave instrument at 120 °C, 250 W. After 10 min, ³¹P NMR analysis showed disappearance of the azaylide with formation of triphenylphosphine oxide. The samples were concentrated, taken up in a small amount of acetonitrile and loaded onto a fluorous SPE column.⁹ First pass elution with 70/30 acetonitrile water provided an organic fraction containing triphenylphosphine oxide and unreacted triphenylphosphine, which was discarded. Second pass elution with THF provided the derived amines.

Without further characterization, each of the three intermediate amines was divided into three equal fractions, which were coupled in parallel with each of the three azido acids 2a–c (1.2 equiv). Couplings were effected in dichloromethane with EDCI (1.2 equiv) and DMAP (0.5 equiv). EDCI was chosen because both it and its derived urea are relatively polar and elute very quickly under the first pass conditions of the fluorous solid phase extraction. The progress of the nine reactions was followed by tlc, and after about 30 min, the solvent was removed and the crude products were subjected to f-SPE as above. Again the first pass fraction was discarded and the second pass fraction (THF) was concentrated to provide the nine coupled azido β-peptides 5aa–5cc, whose complete structures are shown in Figure 2. Yields of these crude products ranged from 55–86% over the two steps, and they all exhibited satisfactory ¹H and ¹³C NMR spectra. Analyses by LCMS also showed the expected molecular weights (see Supplementary Material).

Figure 2.

Structures of the nine dipeptide azides 5

The nine products 5 were then subjected to a second cycle without further purification. First, reduction with triphenylphosphine and f-SPE provided nine amino di-β-peptides (80–100%). These were each divided into three equal portions followed by coupling with the three azido acids 2a–c as above. Evaporation of the THF fraction following f-SPE provided the 27 azido tri-β-peptides 6aaa–6ccc in the yields recorded in Table 1. Also shown in Table 1 is one representative structure (6abc); the full complement of structures are in the Supplementary Material. LCMS analysis of all 27 samples showed the expected molecular weights for the products 6, whose purities were typically 90% or better. Five of the samples selected by molecular weight (including highest, median, lowest) where also analyzed by ¹H and ¹³C NMR to provide additional support for structure and purity.

Table 1.

Yields and MS data for protected azides 6 and final tri-β-peptides 7

entry	% 6	MS 6 (calc/obs)a	% 7 fspeb	% 7 hplcc	MS 7 (calc/obs)a
aaa	33	1177.4/1178.2	92	–d	585.4/586.3
aab	37	1149.4/1150.0	89	–d	557.3/558.3
aac	55	1129.4/1130.2	80	76	537.4/538.3
aba	100	1149.4/1150.2	84	94	557.3/558.3
abb	99	1121.3/1122.0	71	–d	529.3/530.2
abc	94	1101.4/1102.2	64	45	509.3/510.3
aca	58	1129.4/1130.2	61	80	537.4/538.3
acb	74	1101.4/1102.2	72	67	509.3/510.2
acc	41	1081.4/1082.2	68	–d	489.4/490.4
baa	95	1149.4/1150.0	80	84	557.3/558.3
bab	84	1121.3/1122.2	70	94	529.3/530.2
bac	97	1101.4/1102.2	97	93	509.3/510.2
bba	84	1121.3/1122.2	51	99	529.3/530.2
bbb	87	1093.3/1094.2	60	–d	501.3/502.2
bbc	75	1073.3/1074.2	88	86	481.3/482.2
bca	30	1101.4/1102.2	82	64	509.3/537.3e
bcb	46	1073.3/1074.2	76	53	481.3/482.3
bcc	41	1053.4/1054.2	79	87	461.3/462.3
caa	42	1129.4/1130.2	87	91	537.4/538.3
cab	53	1101.4/1102.2	71	75	509.3/510.4
cac	69	1081.4/1082.2	55	78	489.4/490.3
cba	97	1101.4/1102.2	69	71	509.3/510.2
cbb	92	1073.3/1074.2	88	90	481.3/482.2
cbc	88	1053.4/1054.2	88	57	461.3/462.3
cca	37	1081.4/1082.2	82	40	489.4/490.4
ccb	38	1053.4/1054.2	97	83	461.3/469.2e
ccc	33	1033.4/1034.2	78	88	441.4/442.4

^am/z for M + 1 ion;

^byield 7 after fluorous solid phase extraction;

^crecovery of 7 after hplc purification;

^dthe product after f-SPE was pure by HPLC analysis and was not further purified;

^ethe mass spectrum is not consistent with the target product (see text).

Finally, the terminal azides 6 were reduced and the ^FPMB groups were removed simultaneously by reduction with hydrogen in t-butanol catalyzed by palladium hydroxide. Three initial reactions were conducted in vials and the crude products processed by f-SPE in cartridges as above to remove both the catalyst and the residual fluorous tag. This time, the first pass fraction was concentrated and the second pass fraction was discarded. The remaining 24 samples were then hydrogenated together in a 24-well parallel synthesizer, followed by plate-to-plate solid phase extraction¹² and concentration.

After a preliminary analysis by LCMS (reverse phase conditions), most of the crude tri-β-peptides 7 were purified by serial reverse phase HPLC under a standard set of conditions to provide purified products in the yields indicated in Table 1 (see Supplementary Material for all structures). These zwitterionic products are generally white powders that were sparingly soluble in most organic solvents tested. They were then reanalyzed by LCMS to confirm identity and purity. Twenty-five of the products exhibited essentially a single peak with the expected mass. Several of these products were again checked by ¹H NMR analysis (D₂O/CD₃CN) to confirm structure and purity (see Supplementary Material).

Two of the products do not appear to have the expected structures based on MS analysis. The product 7bca exhibited peaks for (M – H₂O + Na) and (M – H₂O + 2Na), suggesting that it might be a dehydrated cyclic peptide. The MS for 7ccb suggests that this product results from reductive deamination (hydrogenolysis) of the terminal benzylic amino group. These side reactions seem to be sequence specific, since they were not observed for any other members of the library.

3. Summary and Conclusions

In summary, we have parlayed three readily available azido acids 2a–c into a small library of tri-β-peptides 7 in amino acid form by fluorous tagging, two cycles of azide reduction and peptide coupling, and detagging with concomitant azide reduction. To expedite the synthesis, intermediates were purified only by fluorous solid phase extraction; however, the final products were purified by preparative hplc to ensure good quality. This approach provided 25 of the 27 library members in acceptable yields and excellent purities.

The results suggest that the azido acid approach to making β-peptide oligomers deserves serious consideration as an alternative to the more traditional amino acid approach. The usefulness of the new approach will grow as a function of the availability of isomerically pure azido acids. The results further show the generality of fluorous solid phase extraction, whether in cartridges or in plate-to-plate format, as a method for rapid yet effective purification of molecules bearing light fluorous tags.

4. Experimental

General procedure 1: S_N2 addition of NaN₃ to β-lactones.^3,5a

The β-lactone (6.0 mmol) was added via syringe to a solution of NaN₃ (12.0 mmol) in anhydrous DMF (35 mL, 0.3 M in lactone) at 50 °C. The resulting homogeneous solution was stirred for 4–5 h. The reaction mixture was cooled to ambient temperature (25 °C), and saturated aqueous NaHCO₃ (30 mL) was added. The resulting heterogeneous mixture was diluted with water until all the precipitated salts dissolved. The resulting mixture was washed with ethyl acetate (2 × 50 mL) and the aqueous layer was acidified with 1 M aqueous HCl to pH ≈ 1. The acidic aqueous layer was extracted with ethyl acetate (3 × 50 mL) and the combined organic portions were washed with water (2 × 50 mL) and brine (2 × 50 mL). The organic portion was dried (Na₂SO₄) and evaporated in vacuo to afford the β-azido acid.

General procedure 2: ^FPMB tagging

To a solution of β-azido acid (0.2 mmol) in CH₂Cl₂ (10 mL) was added N,N-dimethylaminopyridine (DMAP) (12 mg, 0.10 mmol). With stirring, ^FPMB-OH (16, 140 mg, 0.240 mmol) was added to the solution followed by 1-ethyl-3-(3′-dimethylaminopropyl) carbodiimide (EDCI) (46 mg, 0.24 mmol). After 30 min, the mixture was partitioned between Et₂O (30 mL) and 1M aqueous HCl (15 mL). The organic layer was separated and washed with H₂O (10 mL) and brine (10 mL), then dried with Na₂SO₄ and evaporated in vacuo to afford the crude ^FPMB ester. This was purified by flash chromatography (4:1 Hex:EtOAc).

General procedure 3: Staudinger reaction.¹⁰

In a microwave tube, the azido ester or the azido β-peptide (0.2 mmol) was dissolved in dry THF (0.5 mL). A solution of triphenylphosphine (63 mg, 0.24 mmol) in dry THF (1 mL) was added into the microwave tube by syringe. The mixture was heated in the microwave reactor under stirring at 120 °C, 250 W, for 5 min. H₂O (72 mg, 4.0 mmol) was added to the microwave tube via syringe. The resulting mixture was microwaved for 10 min, at 120 °C, 250 W. The reaction progress can be monitored by ³¹P NMR if desired (³¹P NMR spectra were recorded at 121.5 MHz. THF-d₈ was used as solvent. For calibration, the chemical shift of triphenylphosphine was set at −4.80 ppm. The chemical shifts of triphenylphosphine oxide and phosphorus ylide are around 24 ppm and 1 ppm, respectively.).¹³ The solvent was removed in vacuo. A new f-SPE cartridge (5 g) was washed with THF (20 mL) under a vacuum on the SPE manifo ld and preconditioned by passing through 70:30 MeCN:H₂O (30 mL). The crude product was dissolved in MeCN (1 mL) and loaded onto the cartridge using vacuum to ensure that the sample was completely adsorbed onto the silica. The cartridge was washed with 70:30 MeCN:H₂O (50 mL) to obtain the fraction containing the organic compounds, and washed with THF (30 mL) to obtain the fraction containing the fluorous compounds. The fluorous fraction was dried with Na₂SO₄ and the solvent was evaporated in vacuo to afford the amine.

General procedure 4: amide coupling

DMAP (12 mg, 0.10 mmol) was added to a solution of amine (0.2 mmol) in CH₂Cl₂ (2 mL). With stirring, β-azido acid (0.24 mmol) was added to the solution, followed by adding EDCI (46 mg, 0.24 mmol) and CH₂Cl₂ (1 mL). The mixture was stirred for 30 min, and then the solvent was removed in vacuo. A new f-SPE cartridge (5 g) was washed with THF (20 mL) under a vacuum on the SPE manifold and preconditioned by passing through 70:30 MeCN:H₂O (30 mL). The crude product was dissolved in MeCN (1 mL) and loaded onto the cartridge using vacuum to ensure that the sample was completely adsorbed onto the silica. The cartridge was washed with 70:30 MeCN:H₂O (50 mL) to obtain the fraction containing the organic compounds, and washed with THF (30 mL) to obtain the fraction containing the fluorous compounds. The fluorous fraction was dried with Na₂SO₄ and the solvent was evaporated in vacuo to afford the azido β-peptide.

General procedure 5: hydrogenation

In a GreenHouse Classic Parallel Synthesizer, a solution of each azido-tripeptide (8 mg) in tert-butanol (2 mL) was loaded to each test tube. Pd(OH)₂/C (2 mg, 25 w%) was added to each solution. The apparatus was evacuated and then filled with hydrogen gas by a balloon. With stirring on, all reactions were done in 24 h according to TLC. The solvent was removed in vacuo using a vacuum centrifuge. The 24 reaction mixtures were directly loaded onto a preconditioned SPE cartridge plate, with each cartridge packed with FluoroFlash^™ silica gel (3 g).¹⁴ The cartridges were first eluted with 70:30 MeCN:H₂O (2 × 5 mL). The solvent was removed in vacuo by vacuum centrifuge to afford the β-tripeptides. The cartridges were washed with THF (3 × 5 mL) to remove the ^FPMB residue and they were then ready for reuse.

Compound Characterization

Complete characterization data, including copies of NMR spectra and HPLC traces, are provided in the Supplementary Material.