Amide bond formation by using amino acid fluorides

Amino acid fluorides have been used extensively in peptide synthesis. The following review surveys the formation and coupling of amino acid fluorides in both peptide and organic synthesis.


Introduction
N-Protected amino acid chlorides have been known since the early 1900's. 1 They have been rarely used in peptide synthesis until recently when they have been successfully applied to the rapid coupling of Fmoc-amino acids. 2,3However, because of their high reactivity and sensitivity to hydrolysis, acid chlorides should be prepared from the parent amino acids immediately before use 2 and a large excess of reactant is typically required for their synthesis. 3Acid fluorides, on the other hand, are known to be more stable to hydrolysis than acid chlorides 4 but they have been rarely used in organic synthesis because of their presumed low reactivity toward common nucleophiles. 3However, fluorides like indole 2-carbonyl fluoride have been shown to react readily with silylated aliphatic and aromatic amines. 3,5All abbreviations not cited in the text are listed in the Appendix.)

Formation of Amino Acid Fluorides
Amino acid fluorides (AA-F) can be synthesized using two main methods as outlined below.
The first method uses cyanuric fluoride (2) as the fluorinating agent (Scheme 1).In a typical experiment, equimolar amounts of the amino acid 1, pyridine and cyanuric fluoride (2) are mixed and stirred for 3-4 hours in dichloromethane at room temperature. 3At that time, ice-water is added to the reaction mixture and the precipitated cyanuric acid ( 4) is filtered off. 3 The organic phase is evaporated to dryness, which generally affords the pure amino acid fluoride 3 in crystalline form. 3

Scheme 1
The second method uses TFFH, 5 as the fluorinating agent (Scheme 2).TFFH is a nonhygroscopic air stable salt, that can be handled under routine conditions, 6 and is available commercially.Infrared examination shows that in the presence of DIPEA, Fmoc-amino acids are converted using TFFH to the acid fluorides 3. 6 In dichloromethane solution at room temperature, an IR absorption characteristic of the carbonyl fluoride moiety (1842 cm -1 ), appears after about 3 min, with complete conversion to the acid fluoride occurring after 8-15 min. 5For hindered amino acids (e.g., Aib) complete conversion may require 1-2 h. 6If desired, the acid fluorides may be isolated and purified, making TFFH a benign substitute for the corrosive cyanuric fluoride.Another reagent which uses the same methodology as shown above is BTFFH (Figure 1).BTFFH behaves the same as TFFH in its ability to form amino acid fluorides in both the solution and solid phase. 7The main advantage in using BTFFH over TFFH is that one does not form volatile or toxic byproducts. 7,8urthermore, additives such as PTF and pyridine-hydrogen fluoride reagent have been used successfully to obtain amino acid fluorides. 9

N
F N PF 6

Figure 1
Protecting groups such as Fmoc, Boc, and Z can be used on the α-nitrogen of the amino acid en route to the corresponding amino acid fluorides. 10Generally the corresponding acid chlorides bearing the Boc or t-butyl protecting groups were either too sensitive to be isolated or were subject to facile degradation on storage. 10Z-protected amino acid chlorides have also proven not to be stable, undergoing both hydrolysis and conversion to the corresponding Leuch's anhydrides 10 thereby limiting their synthetic utility. 10New in situ methods for the preparation of Z amino acid chlorides have been described in response to the above problems. 10The α-Fmoc, α-Boc, and α-Z amino acid fluorides can be synthesized, purified, and stored for extended periods without experiencing hydrolysis. 10

Solid-Phase and Solution-Phase Couplings
Amino acid fluoride couplings have been effective in both solution and solid-phase arenas.Both methods have an absence of appreciable racemization (<1%) 3,10,11 and are normally rapid (<20 min) reactions. 3,10,1111b Due to the nature of the C-F bond, acyl fluorides are of greater stability than the corresponding chlorides toward neutral oxygen nucleophiles such as water or methanol yet appear to be of equal or nearly equal reactivity toward anionic nucleophiles and amines. 3,10,11olution-phase couplings can be carried out using either of the two methods discussed below: 1. Boc-AA-F (1.1 mmol) in 10 mL of CH 2 Cl 2 was added over a period of 60 seconds to a stirred solution of H-AA-OEt•HCl (1 mmol) in 10 mL of H 2 O containing NaHCO 3 (2 mmol).The mixture was stirred for 20 min (after 5-10 min IR examination showed the acid fluoride band 1845 cm -1 to have essentially disappeared).After washing of the CH 2 Cl 2 solution twice each with 5% HCl, 10% NaHCO 3 , and H 2 O, the dried solution was evaporated in vacuo and the crude dipeptide was recrystallized from ether/hexane to give the pure dipeptide.[Yields were typically in the 65-90% range.] 10 2. One phase couplings were carried out by addition of the acid fluoride in dry CH 2 Cl 2 to a solution of the amino acid ester hydrochloride and 2 equivalents of diisopropylethylamine or N-methylmorpholine in CH 2 Cl 2 over a period of 60 s.Workup followed the discussion above.[Yields were typically in the 75-90% range.] 10 Acid fluoride couplings have also been applied to solid-phase synthesis.In earlier work the AA-F was synthesized and then used on the solid-phase system.In recent years, the method of choice for solid-phase peptide synthesis has been to use TFFH to synthesize the AA-F in situ.TFFH is an ideal coupling reagent for solid phase syntheses, being readily available, inexpensive, and capable of providing crude peptides of high quality.11b An example is the assembly of peptide 7, which, due to the difficult Aib-Aib coupling, has previously 11 been used to demonstrate the superiority of HATU over HBTU (Figure 2).11b H-Tyr-Aib-Aib-Phe-Leu-NH 2

Figure 2
Using DMF as solvent and a Biosearch 9050 instrument programmed for 7-min preactivation, 7-min deblocking, and 30-min coupling [5-fold excess of acid, 10-fold excess of base (DIPEA)] for all amino acids except Aib-Aib, for which a 1-h double coupling was used, pentapeptide 7 was obtained in 88% yield; purity of crude product, 92%; amount of des-Aib tetrapeptide, 4%.11b By contrast, under similar conditions the earlier syntheses 12 HATU gave, 94% purity, and HBTU, 43% purity.11b Fmoc-amino acid fluorides have been used extensively on the solid-phase system, 3,11 and have even been used for Multiple Peptide Synthesis (MPS) 13 due to their exceptional reactivity, high solubility in organic solvents (>1 M in DMF), and stability in solution over extended periods of time. 14

Hindered Amide Couplings
Fmoc-amino acid fluorides have been shown to be excellent coupling reagents for both solution and solid-phase peptide syntheses 10a,15a and for the efficient acylation of hydroxyl moieties. 16,18 wever, their most impressive application is the coupling of adjacent sterically-hindered amino acids such as Aib as demonstrated by the first successful solid-phase synthesis of the peptaibols, 17 naturally occurring peptides containing a high content (up to 60%) of Aib residues. 18he couplings of acid fluorides derived from Aib and NMe-Gly have been shown to proceed without difficulty, although problems arose when more hindered substrates were used such a Iva, NMeVal, Deg, and NMe-Aib.[Conditions: 0.5 mmol HCl•AibOMe, 0.55 mmol Fmoc amino acid fluoride, 1.05 mmol DIEA, 5 mL DMF] 18 When the coupling rates were low, premature deblocking of the Fmoc group was observed as a prominent side reaction. 18In addition, IR studies revealed that Fmoc-amino acid fluorides derived from α,α-dialkylated species are converted slowly into the corresponding oxazolones 9 (Scheme 3) when tertiary amines are present.10a,18,19

Scheme 3
It has been reported that Fmoc-amino acid fluorides could be coupled in the absence of base. 18,20Furthermore, an even more effective approach involves the prior treatment of the amino component of the coupling system with a silylating agent such as N,O-bis(trimethylsilyl) acetamide (BSA). 18Amide bonds can be formed readily under mild conditions by reaction of Nsilylamines with acyl fluorides, 18,21 even in the case of sterically hindered secondary amines. 18,22y treating the free amine portion of resulting peptide with BSA overnight in CH 2 Cl 2 prior to adding the amino acid fluoride one is able to effectively couple to highly hindered residues. 18owever, another report states that although Fmoc amino acid fluorides are excellent reagents for coupling moderately hindered amino acids (e.g., Aib-to-Aib) they are not suited for significantly more hindered systems (e.g., Aib-to-MeAib). 23Coupling to the amino group of Nmethylaminoisobutyric acid is at least an order of magnitude more difficult than coupling to its carboxyl function. 23,24While urethane-protected acid chlorides are inherently more reactive than the fluorides they are also ineffective for hindered systems due to complex oxazolone formation. 23This limitation is bypassed if urethane protection is replaced by arenesulfonyl protection and the Aib-to-MeAib and even MeAib-to-MeAib couplings are easily achieved via the appropriate acid chlorides but not the acid fluorides. 23

Scheme 4
The β-cyanoethyl group proved stable during the reaction of amino acid fluoride 11 with protected dinucleoside phosphate 10 providing compound 12 in good yield.25a As a result, any ribo-and deoxyribooligonucleotide, protected on the bases with Fmoc, and on the phosphates with the β-cyanoethyl group, could be easily aminoacylated.25a Amino acid fluorides have also been used in glycopeptide synthesis. 26Common to all types of N-linked glycoproteins is the presence of a β-configurated N-glycosidic linkage between asparagine (Asn) and N-acetylglucosamine (GlcNAc) residues.26a This linkage was formed using two methods.
The first incorporated GlcNAc derived azide 13, which was reacted with properly protected fluorides, in the presence of Lindlar's catalyst and Me 3 SiOMe (to trap HF) under H 2 to afford the desired glyco-amino acid products 15 in 88-93% yield (Scheme 5).26a The second method used a Boc-protected glycosylamine derivative 16, which was converted to the corresponding silyl carbamate 17, and then reacted with the desired amino acid fluoride in the presence of a catalytic amount of tetrabutylammonium fluoride to afford glycol-amino acid 15 in 78-87% yield.26a The above methods were applied to more functionalized systems using trisaccharide systems with similar success.