Method of peptide synthesis

Details Method of peptide synthesis Method of peptide synthesis

1998-11-25

2001-03-20

Celsa, Bennett (Department: 1654)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Synthesis of peptides

C530S333000, C530S338000, C530S339000, C530S340000, C530S341000, C560S161000, C544S235000, C544S253000, C544S262000, C544S334000

Reexamination Certificate

active

06204361

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a new process for effecting peptide synthesis. More specifically, it relates to a new process for preparing an amino acid fluoride, and also to the use thereof in peptide synthesis. Furthermore, the present invention relates to new reagents for preparing the amino acid fluorides.
2. Background of the Invention
Amino acid fluorides have been shown to be a convenient and highly efficient reagent for both solution and solid phase peptide synthesis. When used in peptide synthesis, the peptide formed therefrom is produced in high yield and relatively pure form, with minimal racemization. Furthermore, it has been shown that there are many other advantages associated with the use of amino acid fluorides in peptide synthesis. For example, the acid fluorides allow the syntheses of peptides which incorporate highly-hindered amino acids, such as &agr;-amino isobutyric acid (Aib) and &agr;-ethylalanine (e.g., isovaline, Iva), and the like. Furthermore, amino acid fluorides exhibit several advantages relative to other amino acid halides, e.g., amino acid chlorides or bromides, in the coupling reaction and formation of peptides. For example, unlike the other amino acid halides, amino acid fluorides can accommodate t-butyl side chain protecting groups. Moreover, conversion to the corresponding oxazolone in the presence of t-organic base does not occur, thus avoiding the danger of racemization. Furthermore, the coupling reactions occur readily in the complete absence of an organic base, again avoiding possible racemization.
Moreover, another advantage of amino acid fluorides is that they are easily synthesized from the corresponding amino acid and are isolable in crystalline form. They are generally stable and have a long shelf life.
In view of these advantages, it is highly desirable to utilize amino acid fluorides in peptide synthesis. Thus, peptide synthesis can be effected by first preparing a N-&agr;-amino protected amino acid fluoride and then utilizing this amino acid fluoride as a coupling agent to produce the desired peptide.
Unfortunately, when employing FMOC amino acid fluorides in practical peptide synthesis, difficulties were encountered in the case of two amino acids, arginine and histidine. In the latter case, while FMOC—His (Trt)—F has been synthesized and used in coupling reactions, its long term shelf stability is in doubt. For sulfonamide-protected arginine derivatives (e.g., FMOC—Arg (Pbf)—OH or FMOC—Arg—(Pmc)—OH)), the corresponding acid fluorides could not be synthesized due to their facile cyclization to the corresponding lactam.
Thus, investigations were conducted to improve the efficiency thereof and to overcome these problems. It was felt that the efficiency of the overall process would be enhanced if the amino acid fluoride were produced in situ. Thus, Carpino et al., as described in
JACS
95, 117, 5401, developed a new uronium-style reagent TFFH, 1, which
has been shown to act as a coupling reagent which acts via in situ conversion to an acid fluoride:
Although a useful technique, especially since TFFH is relatively inexpensive, a disadvantage of this method is the need to use a basic reagent, such as N,N-diisopropylethylamine (DIEA) in the activation step (Eq. 1). Indeed, the speed of conversion of the acid to the acid fluoride increases with the number of equivalents of DIEA used (1 eq<<2 eqs<3 eqs<4 eqs).
Although this technique was more efficient than methods heretofore used in peptide coupling, scientific investigations were conducted to improve upon this reagent. It was believed that the in situ process for the preparation of protected amino acid fluorides and the overall process of peptide synthesis would be improved if a method could be found to generate amino acid fluorides in situ without the presence of a basic reagent. The present inventors have found such a method. Moreover, the present in situ process overcomes the difficulties discussed hereinabove with histidine and arginine.
SUMMARY OF THE INVENTION
Accordingly, the present invention is directed to a process of preparing a N-&agr;-amino protected amino acid fluoride in situ by reacting a N-&agr;-amino protected amino acid or acylating derivative thereof in the presence of a coupling agent with an ionic fluoride salt. In a preferred embodiment, the anion has the formula:
Ly(HF)
z
F
−
or TG
1
G
2
G
3
F
2
−
  I
wherein
z is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;
y is 0 or 1;
L is TG
1
G
2
G
3
G
4
;
T is a Group IV element consisting of Si,
Ge, Sn and Pb;
G
1
, G
2
, G
3
and G
4
are independently halogen, hydrogen, alkyl, aryl, aryl alkyl, cycloalkyl or cycloalkyl alkyl.
The present invention is also directed to the preparation of peptides from the in situ preparation of acid fluorides in accordance with the procedure described hereinabove. Finally, the present invention is directed to the novel fluoride salts useful in the in situ preparation of amino acid fluorides.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
As indicated hereinabove, an aspect of the present invention is directed to the in situ synthesis of an N-&agr;-amino protected amino acid fluoride from an N-&agr;-amino protected amino acid or acylating derivative thereof and an ionic fluoride salt in the presence of a coupling agent.
The term “amino acid” is a term of art that is readily understood by the skilled artisan.
As used herein, the term “amino acid” refers to an organic acid containing both a basic amino group (NH
2
) and an acidic carboxyl group (COOH). Therefore, said molecule is amphoteric and exists in aqueous solution as dipole ions. (See, “The Condensed Chemical Dictionary”, 10th ed. edited by Gessner G. Hawley, Van Nostrand Reinhold Company, London, Eng. p.48 (1981)). The preferred amino acids are the &agr;-amino acids. They include, but are not limited to the 25 amino acids that have been established as protein constituents. They must contain at least one carboxyl group and one primary or secondary amino group on the amino acid molecule. They include the naturally occurring amino acids. For example, they include such proteinogenic amino acids as alanine, valine, leucine, isoleucine, norleucine, proline, hydroxyproline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, hydroxylysine, ornithine, arginine, histidine, naphthylalanine, penicillamine, &bgr;-alanine, isovaline, &agr;-amino isobutyric acid, and the like.
A N-&agr;-amino protected amino acid designates an amino acid in which the &agr;-amino group contains a blocking group. These type of blocking groups (also designated as protecting groups) are well known in the art. Examples include 9-fluorenylmethyloxy-carbonyl(FMOC), 2-chloro-1-indanyl methoxy carbonyl (CLIMOC), and benz-[f]-indene-3-methyloxycarbonyl (BIMOC) and dbd-TMOC which are discussed in U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396, 4,581,167, 4,394,519, 4,460,501 and 4,108,846, and the contents thereof are incorporated herein by reference as if fully set forth herein. Moreover, other amino protecting groups such as 2-(t-butyl sulfonyl)-2-propenyloxycarbonyl (Bspoc) and benzothiophene sulfone-2-methoxycarbonyl (Bsmoc) are discussed in copending application, U.S. patent application Ser. No. 364,662 and the subject matter therein is incorporated herein by reference. Other amino protecting groups include those described in an article entitled “Solid Phase Peptide Synthesis” by G. Barany and R. B. Merifield in
Peptides,
Vol. 2, edited by E. Gross and J. Meienhoffer, Academic Press, New York, N.Y., pp. 100-118 (1980), the contents of which are incorporated herein by reference. These N-amino protecting groups include such groups as FMOC, Bspoc, Bsmoc, t-butyloxycarbonyl (BOC), t-amyloxycarbonyl (Aoc), &bgr;-trimethylethyloxycarbonyl (TEOC), adamantyloxycarbonyl (Adoc), 1-methylcyclobutyloxycarbonyl (Mcb), 2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc), 2-(p-phenylazopheny

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