Selective acylation of epsilon-amino groups

Details Selective acylation of epsilon-amino groups Selective acylation of epsilon-amino groups

1999-07-08

2003-01-28

Riley, Jezia (Department: 1656)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Insulin; related peptides

C514S003100

Reissue Patent

active

RE037971

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to the acylation of proteins. More particularly, the invention relates to a one-step process for selectively acylating the &egr;-amino group of proinsulin, insulin or an insulin analog in the presence of a free &agr;-amino group.
BACKGROUND OF THE INVENTION
The acylation of amino groups is one of the most common means employed for chemically modifying proteins. General methods of acylation are set forth in Methods of Enzymology, 25:494-499 (1972) and include the use of activated esters, acid halides, or acid anhydrides. The use of activated esters, in particular N-hydroxysuccinimide esters of fatty acids is a particularly advantageous means of acylating a free amino acid with a fatty acid. Lapidot et al., J. of Lipid Res. 8:142-145 (1967). Lapidot et al. describe the preparation of N-hydroxysuccinimide esters and their use in the preparation of N-lauroyl-glycine, N-lauroyl-L-serine, and N-lauroyl-L-glutamic acid.
Early studies of selectively acylating the amino groups of insulin are described in Lindsay et al., in Biochem. J. 121:737-745 (1971). Lindsay et al., describe the reactivity of insulin with N-succinimidyl acetate at low reagent concentration and near neutral pH as producing two mono-substituted products. Phe
B1
-acetyl-insulin and Gly
A1
-acetyl insulin. At pH 8.5, the amount of Phe
B1
-acetyl insulin produced is lowered and Lys
B29
-acetyl-insulin is also produced. Thus, Lindsay et al., conclude at pH 6.9 the order of reactivity is Glycine(A1)≈Phenylalanine(B1)>>Lysine (B29) and at pH 8.5 Glycine(A1)>Phenylalanine=Lysine (B29). Id.
Lindsay et al., U.S. Pat. No. 3,869,437, disclose the acylation of the B
1
amino acid with an acyl group containing up to seven carbons and optionally blocking the A
1
- and/or B
29
-amino group with an acyl group with up to four carbons. N-hydroxysuccinimide esters are described as particularly advantageous acylating agents. In order to produce the maximum yield of insulin acylated at the B
1
-amino group, the proportion of acylating agent is relatively low (one to not more than two molar equivalents of acylating agent). In addition, the maximum yield of mono-substituted B
1
product is produced at a pH at or near about pH 7. At pH 8.5 to 9.0, the yield of the desired B
1
acylated product falls off considerably in favor of additional substitution at positions A
1
and B
29
.
D. G. Smyth, in U.S. Pat. No. 3,868,356 and Smyth et al., in U.S. Pat. No. 3,868,357 disclose N-acylated, O-substituted insulin derivatives in which at least one of the A
1
, B
1
or B
29
amino acid amino groups is converted into a blocked amino group. The acylation is carried out with a relatively small excess of acylating agent, e.g., from 2 to 3 moles per amino group at a neutral or mildly alkaline pH, e.g., 7-8. The reaction proceeds in very high yield with the formation of the di-substituted derivative resulting from the reaction of the A
1
- and B
1
-amino groups. In the presence of excess acylating agent, e.g., up to 10 molar, the reaction proceeds additionally at the B
29
-amino group to form the tri-substituted derivative.
To selectively acylate insulin, Muranishi and Kiso, in Japanese Patent Application 1-254,699, disclose a five-step synthesis for preparing fatty acid insulin derivatives. Step one, the activated fatty acid ester is prepared; Step two, the amino groups of insulin are protected with p-methoxy benzoxy carbonylazide (pMZ); Step three, the insulin-pMZ is reacted with the fatty acid ester; Step four, the acylated insulin is deprotected; and Step five, the acylated insulin is isolated and purified. Most notably, selective acylation of one amino group is only achieved by using the pMZ blocking group to protect the other amino groups. Using this methodology, Muranishi and Kiso prepare the following compounds: Lys
B29
-palmitoyl insulin (the &egr;-amino group is acylated), Phe
B1
-palmitoyl insulin (the N terminal &agr;-amino group of the B chain is acylated), and Phe
B1
, Lys
B29
-dipalmitoyl insulin (both the &egr;-amino and the N-terminal &agr;-amino group are acylated).
Similarly, Hashimoto et al., in Pharmaceutical Research 6:171-176 (1989), teach a four step synthesis for preparing N-palmitolyl insulin. The synthesis includes protecting and deprotecting the N-terminal. A
1
-glycine and the &egr;-amino group of B
29
-lysine, with pMZ. Under the conditions described in the reference, two major acylated products are prepared, B
1
-mono-palmitoyl insulin and B
1
, B
29
-dipalmitoyl insulin.
Therefore, prior to the present invention, the selective acylation of the B
29
-N
&egr;
-amino group of insulin was carried out by protecting and subsequently deprotecting the &agr;-amino groups. The present invention provides a selective one-step synthesis for acylating the &egr;-amino group of proinsulin, insulin and insulin analogs. It is quite surprising that the invention is able to selectively acylate the &egr;-amino group in a one step process in high yield. Thus, the invention eliminates the need to protect and subsequently deprotect other amino groups of the protein. The invention provides more efficient and less expensive means of preparing &egr;-amino acylated insulin derivatives.
SUMMARY OF THE INVENTION
The present invention provides a process of selectively acylating proinsulin, insulin, or an insulin analog having a free &egr;-amino group and a free &agr;-amino group with a fatty acid, which comprises reacting the &egr;-amino group with a soluble activated fatty acid ester under basic conditions in a polar solvent.


REFERENCES:
patent: 3471464 (1969-10-01), Bellet et al.
patent: 3528960 (1970-09-01), Haas
patent: 3591574 (1971-07-01), Fenichel et al.
patent: 3752798 (1973-08-01), Amird et al.
patent: 3755569 (1973-08-01), Fenichel et al.
patent: 3823125 (1974-07-01), Grant et al.
patent: 3864325 (1975-02-01), Smyth
patent: 3868356 (1975-02-01), Smyth
patent: 3868357 (1975-02-01), Smyth et al.
patent: 3869437 (1975-03-01), Lindsay et al.
patent: 3883496 (1975-05-01), Geiger
patent: 3883500 (1975-05-01), Geiger et al.
patent: 3884897 (1975-05-01), Geiger et al.
patent: 3950517 (1976-04-01), Lindsay et al.
patent: 4013628 (1977-03-01), Obermeier et al.
patent: 4014861 (1977-03-01), Geiger et al.
patent: 4218539 (1980-08-01), Weltman
patent: 5304473 (1994-04-01), Belagaje et al.
patent: 5905140 (1999-05-01), Hansen
patent: 214826 (1986-08-01), None
patent: 383472 (1990-02-01), None
patent: 1260963 (1972-01-01), None
patent: 1415333 (1975-11-01), None
patent: 1492997 (1977-11-01), None
patent: 1-254699 (1989-10-01), None
patent: WO92/01476 (1992-02-01), None
patent: WO95/07931 (1995-03-01), None
Geiger, et al.,Biological Activity of Insulin Analogues Substituted at the Amino Group of B1-Phenylalanine,from Proceedings of the Second International Insulin Symposium, Aachen, Germany, Sep. 4-7, 409-415, (1979).
R. Geiger and R. Obermeier,Contribution of peptide chemistry to our knowledge of insulin and diabetes,from Proceedings of the Symposium on Proinsulin, Insulin and C-Peptide, Tokushima, Jul. 12-14, 62-72, (1978).
Rösen, et al,A1-Modified Insulins: Receptor Binding and Biological Activity,from Proceedings of the Second International Insulin Symposium, Aachen, Germany, Sep. 4-7, 403-408, (1979).
Hashimoto, et al,Pharmaceutical Research,6:2, 171-176, (1989).
Geiger,Chemiker Zeitung,Sonderdruck 100, 111-129, (Translation attached), (1976).
I. MacIntyre and M. Szelke, et al.,Molecular Endocrinology,Proceedings of Endocrinology '77 held at the Royal College of Physicians, London, England on Jul. 11-15, 27-42, (1977).
W. Scheider,Journal of Physical Chemistry,84:8, 925-928, (1980).
D.G. Lindsay and S. Shall,Biochem.J.,115, 587-595, (1969).*
Muranishi, et al.,Journal of Controlled Release,19, 179-188, (1992).*
Hashizume, et al.,J. Pharm. Pharmacol.,44 555-559, (1992).*
Lindsay, et al.,Biochem.J.,121, 737-745, (1971).*
J.F. Riordan and B.L. Vallee,Methods of Enzymology,25, 494-499, (1972).*
Lapidot, et al.,Journal of Lipid Research,8, 142-145, (1967).*
Anderson, et al.,Journal o

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