Synthesis of N-Fmoc-(2S,3S,4R)-3,4-dimethylglutamine. An Application of Lanthanide-Catalyzed Transamidation

Abstract

N-Fmoc-(2S,3S,4R)-3,4-dimethylglutamine (6) was synthesized from t-butyl N-Boc-(2S,3S,4R)-dimethylpyroglutamate (13). This synthesis involved selective deprotection of a Boc group from a lactam nitrogen in the presence of a t-butyl ester, Fmoc protection of the lactam, and a lanthanide-catalyzed transamidation reaction of the Fmoc-protected lactam using ammonia and dimethylaluminum chloride. The scope of Lewis acid-catalyzed transamidation of acylated lactams was explored through the variation of lanthanide, lactam, acyl group, amine and aluminum reagent. The reactivity of various metal triflates was found to vary in the qualitative order: Yb∼Sc>Er∼Eu∼Sm>Ce∼Ag^I>Cu^II∼Zn. Intriguingly, catalysis was only observed when ammonia was the nitrogen nucleophile; addition of other amidoaluminum complexes to acyl lactams was found to be insensitive to the addition of lanthanides.

Introduction

The non-proteinogenic amino acid (2S,3R,4S)-3,4-dimethylglutamine (DiMeGln) has been found to be a key component of the cyclic depsipeptide natural products callipeltin A¹ (1) and papuamides A (2) and B (3).² Callipeltin A (1) was isolated from a shallow water sponge of the genus Callipelta (order Lithistida) by Minale and coworkers and shown to have both antitumor and anti HIV activities.¹ Likewise, the papuamides were isolated from the sponges Theonella mirabilis and Theonella swinhoe by Boyd and coworkers and shown to also possess both antitumor and anti-HIV activities.²

To date, syntheses of DiMeGln have been reported by Joullié,³ Hamada,⁴, and ourselves.⁵ In all three cases, however DiMeGln was synthesized with acid-labile protecting groups on N_α. In our case, an Fmoc-protected version of DiMeGln was desired for an Fmoc-based solid phase synthesis of 1, 2 and related molecules. However, when the Boc-protected intermediate 4 was deprotected and reprotected with an Fmoc group under Schotten-Baumann conditions, it was found that the product 5 was a mixture of C-4 epimers, presumably as a result of acid-catalyzed enolization of the side chain amide (Scheme 1).⁶ Variation of the deprotection conditions failed to alleviate this problem, so an alternate solution was sought.

Scheme 1.

Results and Discussion

Our revised strategy to obtain the Fmoc-protected DiMeGln residue 6 (Scheme 2) called for ammonolysis of an Fmoc-protected lactam 7 that would be synthesized from the previously synthesized intermediate 8 via selective Boc deprotection of the lactam nitrogen in the presence of the t-butyl ester followed by Fmoc protection of the lactam nitrogen.

Scheme 2.

The model compound 9 (Scheme 3) was selected for its ready accessibility from commercially available (S)-pyroglutamic acid.^7,8 Extensive exploration revealed two methods for selective deprotection of the lactam (Scheme 3). In the first method, microwave irradiation of 9 on silica support for 1 minute⁹ gave the desired product 10 in 35-75% yield. Alternatively, catalytic Yb(OTf)₃ in THF effectively deprotected 9 after 18 hours,¹⁰ giving 10 in quantitative yield. Despite the quicker reaction time and easier workup of the microwave deprotection method, deprotection with Yb(OTf)₃ was adopted as the method of choice owing to variability in yields using microwave radiation.

Scheme 3.

Scheme 4.

The next step in the synthesis called for Fmoc protection of a lactam nitrogen. This was accomplished by deprotonation of the amide nitrogen of 10 LHMDS at -78°C, followed by inverse addition of the deprotonated amide into 5 equivalents of Fmoc-Cl (Scheme 4). This procedure gave the desired product, 11, in 76% isolated yield after 14 h.

Scheme 5.

In the next step, 11 was subjected to the ammonolysis conditions that had previously worked successfully with its Boc-protected analogue, 8.⁵ To our surprise, reaction of 11 with AlMe₃-NH₃¹¹ gave only 38% of the desired product 12 and after 2 hours led to substantial deprotection of the Fmoc group. It was inferred from these results that the reagent was basic enough to deprotect an Fmoc carbamate. It was reasoned that, to avoid Fmoc deprotection, a faster or less basic ammonolysis reaction was needed. To this end, the use of Lewis acids as catalysts was investigated. In an early screening experiment, it was discovered that addition of catalytic Yb(OTf)₃ catalyzes ammonolysis of 11 by AlMe₃-NH₃ very efficiently (Scheme 5). After optimization studies it was determined that usage of 6-10 equivalents of AlMe₃, addition of 40 mol% lanthanide catalyst, and changing the solvent from CH₂Cl₂ to THF were needed to obtain high yields.

Scheme 6.

Under these conditions, ammonolysis of 11 was nearly instantaneous even at 25°C. On the basis of these encouraging results, other Lewis acidic metal triflates - Sc(OTf)₃, Er(OTf)₃, Eu(OTf)₃, Sm(OTf)₃ Ce(OTf)₃, Ag(OTf), Cu(OTf)₂, and Zn(OTf)₂ - were also screened as potential catalysts of ammonolysis (Table 1). As expected, Sc(OTf)₃ was as reactive as Yb(OTf)₃ and both proved to be the most active Lewis acid catalysts tested. Although Er(OTf)₃, Eu(OTf)₃ and Sm(OTf)₃ gave slower reactions that went to completion in 5 minutes, reaction with Eu(OTf)₃ and Sm(OTf)₃ resulted in the production of fewer side products. The use of Ce(OTf)₃, Ag(OTf), Cu(OTf)₂ and Zn(OTf)₂ as catalysts resulted in slower reactions that were complete in 20-30 minutes with moderate yields (Table 1). In the best cases, the yield of ammonolysis product was improved and the time of reaction drastically reduced. It was also noted that the amount of Fmoc deprotection observed in these reactions was substantially less than in the uncatalyzed version. Whether the decrease in Fmoc deprotection results from lessened basicity of the reagent or is simply a result of the much shorter reaction times we cannot determine.

Table 1.

Lewis Acid Catalysis of Transamidation

Catalysta	Time, min	Yield, %
-	120	38
Yb(OTf)3	2	87
Sc(OTf)3	2	88
Er(OTf)3	5	78
Eu(OTf)3	5	91
Sm(OTf)3	5	91
Ce(OTf)3	20	70
Ag(OTf)	20	36
Cu(OTf)2	30	24
Zn(OTf)2	30	67

^a40 mol % used.

It has also recently been shown that the use of Me₂AlCl/NH₃ affords greater reactivity than AlMe₃/NH₃ for the ammonolysis of sterically demanding esters.¹² All the metal triflates mentioned above were also screened with Me₂AlCl instead of AlMe₃. Although the reactivity trend was almost the same, and yields were slightly better (see Table 2), reactions with Me₂AlCl were slower their AlMe₃ counterparts.

Table 2.

Lanthanide catalyzed transamidation of Lactams

Amine	Lactam	Al reagent	Catalysta	Time, min	Yield, %
NH3	7	AlMe3	-	120	61
NH3	7	AlMe3	Yb(OTf)3	10	32
NH3	7	Me2AlCl	-	120	25
NH3	7	Me2AlCl	Eu(OTf)3	30	77
NH3	11	AlMe3	-	120	38
NH3	11	AlMe3	Yb(OTf)3	2	87
NH3	11	Me2AlCl	Eu(OTf)3	10	91
NH3	16	AlMe3	-	60	40
NH3	16	Me2AlCl	Eu(OTf)3	60	90
NH3	9	AlMe3	-	10	85
NH3	9	AlMe3	Yb(OTf)3	10	86
PhCH2NH2	11	AlMe3	-	5	80
PhCH2NH2	11	AlMe3	Yb(OTf)3	10	80
PhCH2NH2	11	Me2AlCl	Eu(OTf)3	40	87
Et2NH	11	AlMe3	-	180	b
Et2NH	11	AlMe3	Yb(OTf)3	60	b
t-BuNH2	11	AlMe3	-	360	c
t-BuNH2	11	AlMe3	Yb(OTf)3	360	c

^a40 mol %

^bFmoc deprotection

^cPartial Fmoc deprotection.

With the methodology in hand, our efforts turned to the synthesis of the desired product 6, beginning with the conversion of the previously synthesized intermediate 13 to its t-butyl ester 8 using N,N'-diisopropyl-O-tert-butylisourea.⁵ Selective deprotection of the lactam Boc group with catalytic Yb(OTf)₃ gave the lactam 14, which was then protected with an Fmoc group in high yield. Ring opening of the Fmoc-protected lactam 7 using ammonia in conjunction with Me₂AlCl and Eu(OTf)₃ resulted in the formation of 15 in 77% isolated yield. The target compound 6 was obtained from 15 by treatment with TFA in methylene chloride in high yield. In contrast to our previous efforts, the ¹H-NMR of 6 showed no evidence of epimerization.

With the synthesis of 6 thus completed, it was decided to further investigate the scope of lanthanide catalyzed transamidation by examining the reaction of N-Fmoc-caprolactam (16) in addition to 7 and 11 (Table 2).

Except in the case of 7, lanthanide-catalyzed ammonolysis of Fmoc-protected lactams (7, 11 and 16) gave higher yields and shorter reaction times compared to the uncatalyzed reactions. In addition to the Fmoc-protected lactams, Boc-protected lactam 9, was also subjected to ammonolysis reaction conditions. Interestingly, even with the NH₃ no significant catalytic activity by the addition of metal triflates was observed. Amines other than NH₃ were also tried with 11, but in no case was catalysis by the added lanthanide triflate observed.

Conclusion

In summary, N-Fmoc-(2S,3S,4R)-3,4-dimethylglutamine (6) was synthesized in 57% overall yield in five steps starting from t-butyl N-Boc-(2S,3S,4R)-3,4-dimethylpyroglutamate (13). It was also shown that lanthanide triflates catalyze the ammonolysis of Fmoc-protected lactams in conjunction with AlMe₃ or Me₂AlCl. These conditions offer a way to ammonolyze even sterically hindered, Fmoc-protected lactams. With compound 6 in hand, total syntheses of the callipeltins and papuamides are actively under way.

Experimental Section

General Procedure for Boc deprotection of Lactams with Catalytic Yb(OTf)₃.

To a solution of Boc protected lactam in THF (2 ml) Yb(OTf)₃ (45 mol %) was added. The mixture was stirred at rt for 18 hours and the solvent was removed under reduced pressure. The residue was diluted with CH₂Cl₂ (2 ml) and solution filtered through a fine proze funnel. The filtrate was concentrated under reduced pressure. The crude was purified by flash chromatography on silica gel.

t-butyl (2S,3S,4R)-3,4-dimethylpyroglutamate (14). The general procedure for Boc deprotection of lactam was followed using t-butyl N-Boc-(2S,3S,4R)-3,4-dimethylpyroglutamate (8) (89.3 mg, 0.285 mmol) and Yb(OTf)₃ (70.71 mg, 0.114 mmol). Flash column chromatography with 50% EtOAc/petroleum ether yielded 14 as a colorless, flocculent crystalline solid (57.4 mg, 95%): mp 61°C; IR (NaCl, cm^-1) 3233 (br), 2975, 2934, 2871, 1735, 1709, 1458, 1393, 1219, 1158; ¹H NMR (300 MHz, CDCl₃) δ 5.78 (bs, 1H), 3.67 (d, 1H, 4.7Hz), 2.64-2.53 (m, 2H), 1.49 (s, 9H), 1.16 (d, 3H, 6.74Hz), 1.11 (d, 3H, 7.18Hz) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 179.7, 170.9, 82.4, 61.4, 39.1, 37.8, 28, 14.6, 10.4 ppm; HRMS (CI) calcd for C₁₁H₁₉NO₃ (M+) 214.1443, found 214.1446.

General procedure for the Fmoc protection of lactams.

To a solution of the lactam in THF at -78°C LHMDS (0.95 eq, 1M solution in THF) was added slowly. The resultant pale yellow mixture was stirred at -78°C for 15 minutes, and slowly transferred by cannula to a solution of Fmoc-Cl (5 eq) in THF at -78°C. The reaction was allowed to stir at -78°C for 2 hours, after which it was allowed to rise to room temperature. After 14 hours stirring at room temperature, the reaction was quenched by addition of sat. NH₄Cl (2 ml) and H₂O (1 ml). The solution was extracted with EtOAc and washed with brine. The organic phase was dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by flash column chromatography.

t-butyl N-Fmoc-(2S,3S,4R)-3,4-dimethylpyroglutamate (7). The general procedure for Fmoc protection of lactam was followed using t-butyl (2S,3S,4R)-3,4-dimethylpyroglutamate (14) (27 mg, 0.127 mmol) in THF (2 ml). Flash column chromatography with 20% EtOAc/petroleum ether yielded 7 as a colorless oil (48 mg, 87%): IR (NaCl, cm^-1) 3066, 2977, 2935, 2978, 1798, 1761, 1722, 1478, 1451, 1387, 1369, 1303, 1156; ¹H NMR (300 MHz, CDCl₃) δ 7.75 (d, 4H, 7.6Hz), 7.41-7.24 (m, 4H), 4.55-4.3 (m, 3H), 4.14 (s, 1H), 2.85 (p, 1H, 7Hz), 2.50 (p, 1H, 7Hz), 1.44 (s, 9H), 1.16 (d, 3H, 7Hz), 1.10 (d, 3H, 7Hz) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 174.9, 169.6, 151.9, 143.6 143.4, 141.3, 141.2, 127.8, 127.3, 125.5, 119.9, 82.6, 69, 64.9, 46.7, 40.7, 34.7, 27.9, 15.4, 9.9 ppm; HRMS (ESI) calcd for C₂₆H₂₉NO₅ (Na+) 458.1943, found 458.1945.

t-Butyl N^α-Fmoc-(2S,3S,4R)-3,4-dimethylglutamine (15). To a solution of Me₂AlCl (2.52 ml, 2.52 mmol) in CH₂Cl₂ (4 ml) at -8°C was bubbled gaseous NH₃ for 4 min. The reaction was allowed to stir at -8°C for 15 minutes and allowed to warm to room temperature. Over 30 minutes CH₂Cl₂ was removed using a flow of nitrogen gas to afford a colorless slurry. The residue was added THF (7 ml), and let it stir at 25°C for a minute. Then lactam 7 (109.8 mg, 0.252 mmol) and Eu(OTf)₃ (60.4 mg, 0.1008 mmol) in THF (1.1 ml) was added. After 30 min reaction was quenched using 0.1 N HCl (25.2 ml, 2.52 mmol) and EtOAc (10 ml). The solution was then extracted with EtOAc (3×10 ml). Organic fractions were combined, washed with brine (3×10 ml) and dried over Na₂SO₄. After removal of solvent under reduced pressure the crude product was purified by flash column chromatography with 50% EtOAc/hexane followed by 80% EtOAc/hexane yielded 15 as a colorless oil (88 mg, 77%): IR (NaCl, cm^-1) 3345 (br), 3203, 3066, 2975, 2936, 1716, 1662, 1516, 1452, 1427, 1369, 1345, 1156; ¹H NMR (300 MHz, CDCl₃) δ 7.79-7.76 (m, 2H), 7.60 (d, 2H, 7Hz), 7.45-7.27 (m, 4H), 6.83 (br, 1H), 5.64 (d, 1H, 8.8Hz), 5.57 (br, 1H), 4.60-4.55 (m, 1H), 4.46-4.40 (m, 1H), 4.25-4.17 (m, 1H), 2.37 (dd, 1H, 6.5-3.5Hz), 1.82 (br, 1H), 1.49 (s, 9H), 1.16 (d, 3H, 7Hz), 0.94 (d, 3H, 7Hz) ppm; ¹³C NMR (75 MHz, CDCl₃) δ 175.9, 170.7, 156.8, 143.7, 143.4, 141.4, 141.3, 127.8, 127.1, 125.0, 124.9, 120.0, 119.9, 82.9, 66.9, 57.6, 47.3, 41.6, 40.0, 27.9, 16.1, 12.4 ppm; HRMS (ESI) calcd for C₂₆H₃₂N₂O₅ (Na+) 475.2209, found 475.2213.

N-Fmoc-(2S,3S,4R)-3,4-dimethylglutamine (6). A solution of t-Butyl N-Fmoc-(2S,3S,4R)-3,4-dimethylglutamine (15) (88 mg, 0.195 mmol) in 30%TFA-CH₂Cl₂ (1.5 ml) was allowed to stir at room temperature for 90 min. Removal of solvent and TFA by evaporation under reduced pressure yielded N-Fmoc-(2S,3S,4R)-3,4-dimethylglutamine (6) as a fine, colorless powder (71.7 mg, 93%): mp: decomposition >210°C;IR (NaCl, cm^-1) 3323 (br), 3066, 2923, 2851, 1702, 1671, 1609, 1515, 1450, 1412, 1319, 1227, 1034; ¹H NMR (300 MHz, C₂D₆SO) δ 7.89 (d, 2H, 7.6Hz), 7.75 (d, 2H, 6.5), 7.44-7.33 (4H, m), 6.82 (s, 1H), 4.25 (bs, 3H), 4.15 (m, 1H), 2.34 (t, 1H, 7Hz), 1.97 (bs, 1H), 1.42 (s, 1H), 1.09 (d, 3H, 6.4Hz), 0.87 (d, 3H, 5.9Hz) ppm; ¹³C NMR (75 MHz, C₂D₆SO) δ 181, 174.5, 158.7, 145.5, 145.3, 142.7, 128.3, 126.5, 126.3, 121.2, 121.1, 68.2, 57.4, 48.3, 44.0, 40.5, 16.9, 10.5 ppm; HRMS (ESI) calcd for C₂₂H₂₄N₂O₅ (Na+) 419.1583, found 419.1583.

Figure 1.

Callipeltin A (1).

Figure 2.

Papuamides A (2) and B (3).