Organic compounds -- part of the class 532-570 series – Organic compounds – Four or more ring nitrogens in the bicyclo ring system
Reexamination Certificate
2002-05-21
2004-11-30
Seaman, D. Margaret (Department: 1625)
Organic compounds -- part of the class 532-570 series
Organic compounds
Four or more ring nitrogens in the bicyclo ring system
C544S253000, C544S262000, C544S035000
Reexamination Certificate
active
06825347
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to uronium and immonium salts and their use in effecting the acylation step in amide formation, especially during peptide synthesis.
2. Description of the Prior Art
Polypeptides are useful as medicaments. In recent years, peptides have been found to be useful in combating various diseases, such as cancer, diabetes, plant toxins and the like. Additionally, peptides have shown specific activity as growth promoters, suppressants, antibodies, insecticides, contraceptives, anti-hypertensives, sleep-inducers, anti-depressants, analgesics, etc. The list is long and varied.
As more and more polypeptides become of medicinal importance, there is an increasing incentive to improve the methods by which they may be synthesized. Currently, syntheses of peptides are in solution by classical or various repetitive methods. Alternatively, peptides may be prepared on a solid support (Merrifield method). These are all popular techniques in synthesizing peptides from the coupling of two or more amino acids, in synthesizing larger peptides from the coupling of amino acids with smaller peptides or in the coupling of smaller peptides. Solution methods have the advantage of being easily monitored, allowing purification of intermediates, if necessary, at any stage. A major drawback, however, is the relative slow pace of synthesis, with each step being carried out manually.
The major advantage of the Merrifield method is its easy automation so that unattended, computer-controlled machine synthesis is possible. Unfortunately, the method suffers from an inherent deficiency due to the insoluble nature of the support on which the synthesis proceeds. Unless each acylation step occurs with approximately 100% efficiency, mixtures will inevitably be built up on the polymer. The longer the chain, the greater will be the contamination by undesired side reactions. Side products produced in such reactions remain to contaminate the desired product when it is removed from the polymeric matrix at the end of the cycle. These current techniques are not useful in preparing peptides of greater than 30-40 residues; separation of side products from the desired product becomes increasingly difficult when larger peptides are synthesized.
For very long segments (60 or more amino acids), therefore current methods are not satisfactory. Often, mixtures are obtained of such forbidding complexity that it may be difficult or impossible to isolate the desired peptide.
The problems enumerated hereinabove may be eliminated if the proper derivatives of the underlying amino acids and/or the proper conditions for the coupling reaction could be found. Protecting groups, such as t-butyloxycarbonyl (t-Boc) or N-&agr;-(9-fluorenylmethyl)oxycarbonyl (Fmoc), have been used to minimize side reactions. But, additionally, other aspects of the coupling reaction must also be taken into consideration, such as the peptide coupling additive to be used in the coupling reaction.
Additives generally inhibit side reactions and reduce racemization. Heretofore, the most common peptide coupling additive used during peptide coupling for both solutions and solid phase synthesis is 1-hydroxybenzotriazole (HOBt). This reagent has been used either in combination with a carbodimide or other coupling agent or built into a stand-alone reagent, such as 1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or an analogous uronium salt. HOBt is applicable to both stepwise and segment condensations. However, many cases have been encountered in which HOBt is ineffective, possibly because of stearic effects, or low basicity of the amino component. Especially problematic are segment coupling at amino acid units other than glycine or proline, since the problem of racemization may be severe. The related N-hydroxybenzotriazinone (HOOBt) may provide better protection against racemization, but it is rarely used due to competing side reactions involving ring openings.
Other reagents for facilitating peptide coupling have also been described. For example, in
Tetrahedron Letters,
1994, 35, 2279-2282, Carpino, et al. disclose that 1-hydroxy-7-azabenzotriazole and its corresponding uronium salts, designated HAPyU and AOP were effective in promoting peptide coupling and avoiding racemization in a model solid-phase peptide segment coupling process. In addition, Carpino, et al. disclose in
J. Org. Chem.,
1994, 59, 695-698 that azabenzotriazolyluronium salts, e.g., designated as HBTU, HATU, HBPyU, HAPyU, HBMDU and HAMDU, are useful in peptide synthesis. Other publications such as Ehrlich, et al., disclose that the uronium salts, designated as HAPyU and TAPipU were useful for promoting peptide cyclization with a minimum of racemization.
U.S. Pat. No. 5,644,029 to Carpino discloses among other things, the use of compounds of the following formula in promoting peptide coupling:
and N-oxides thereof and salts thereof wherein
R
1
and R
2
taken together with the carbon atoms to which they are attached form a heteroaryl ring wherein said heteroaryl ring is an oxygen, sulfur or nitrogen containing heteroaromatic containing from 3 and up to a total of 13 ring carbon atoms, said heteroaryl may be unsubstituted or substituted with lower alkyl or an electron-donating group;
Y is O, NR
4,
CR
4
R
5
;
R
5
is independently hydrogen or lower alkyl;
X is CR
6
R
7
or NR
6
;
R
6
and R
7
are independently hydrogen or lower alkyl; or R
6
and R
7
taken together form an oxo group or when n=0, R
4
and R
6
taken together may form a bond between the nitrogen or carbon atom of Y and the nitrogen or carbon atom of X;
Q is (CR
8
R
9
) or (NR
8
);
when n is 1, R
4
and R
8
taken together may form a bond between the ring carbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R
8
;
n is 0, 1 or 2;
R
3
is hydrogen, lower alkyl carbonyl, aryl carbonyl, lower aryl alkyl carbonyl,
a positively charged electron withdrawing group, SO
2
R
14
, or
R
14
is lower alkyl, aryl or lower arylalkyl; q is 0-3;
R
8
and R
9
are independently hydrogen or lower alkyl or R
7
and R
8
taken together with the carbon to which they are attached form an aryl ring, AA
1
is an amino acid and BLK is an amino protecting group, and m is 0 or 1.
It teaches that the compounds are prepared by reacting compounds of the formula:
with R
3
L in the presence of a base under substitution reaction conditions, in which R
1
, R
2
, Y, Q, n, X, and R
3
are as defined hereinabove and L is a leaving group, such as halide.
At the time of the publications of the aforementioned articles as well as of the time of the filing of the aforementioned patent, it was believed that all of the compounds described therein had the formula shown hereinabove wherein the R
3
was bonded to the oxygen atom (the O-isomer). This belief was based upon the structure of the corresponding phosphonium derivatives, such as benzotriazol-1-yl-N-oxy-tris(dimethylamino) phosphonium hexafluorophosphite (BOP)
and benzotriazol-1-yl-N-oxy-tris-(pyrrolidino)-phosphonium hexafluorophosphate
in which the oxygen atom was bonded to the cationic phosphonium group. Based on these structures, when the uronium salt derivatives of hydroxybenzotriazole were first described it was assumed, by the scientific community by analogy, to have the structure hereinbelow:
wherein the positively charged uronium ion was bonded to the oxygen atom.
In addition, when other coupling reagents, such as HATU were described, by analogy to the structures assigned to the hydroxybenzotriazole derivatives, it was also assumed that HATU and its derivatives also had the structure:
In fact, based on the same assumptions, it was believed that the uronium salts in general, for example, described hereinabove in the aforementioned publications had similar structures wherein the oxygen atom was bonded to the positively charged cation. Because such structures were believed to be the O-isomers, x-ray crystallography of these new O-isomers was not performed.
However, finally when x-ray cry
Carpino Louis A.
El-Faham Ayman
Imazumi Hideko
Research Corporation Technologies Inc.
Scully Scott Murphy & Presser
Seaman D. Margaret
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