PNA synthesis using a base-labile amino protecting group

Details PNA synthesis using a base-labile amino protecting group PNA synthesis using a base-labile amino protecting group

2000-02-01

2001-11-13

Low, Christopher S. F. (Department: 1653)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Synthesis of peptides

C562S553000, C562S561000, C530S338000, C536S018700, C544S264000

Reexamination Certificate

active

06316595

ABSTRACT:

Peptide nucleic acids (PNA) are compounds which are analogs of DNA in which the deoxyribose phosphate skeleton has been replaced by a peptide oligomer. The syntheses which have thus far been described in the literature (Michael Egholm, Peter E. Nielsen, Ole Buchardt and Rolf H. Berg, J. Am. Chem. Soc. 1992, 114, 9677-9678; Ole Buchardt, Michael Egholm, Peter E. Nielsen and Rolf E. Berg, WO 92/20702) use the acid-labile tert-butyloxycarbonyl (Boc) protecting group, which is eliminated with medium-strength acids such as, for example, trifluoroacetic acid, for temporarily protecting the amino group of the monomer. Solid phase synthesis of oligomers then follows tho customary peptide synthesis method as described, for example, by Merrifield (B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149). A strong acid, usually liquid hydrogen fluoride, is used in this case to cleave the PNA oligomer from the solid support. As a consequence, these reaction conditions, in particular the repeated treatment with trifluoroacetic acid, do not permit a reaction in open reaction vessels, as is the case, for example, when multiple peptide synthesizers are employed (review: G. Jung and A. Beck-Sickinger, Angew. Chem. 104 (1992) 375-391).
The object of the invention is to develop a synthesis process which uses a base-labile, temporary amino protecting group for constructing the PNA oligomers and which permits cleavage of the oligomer from the solid support using weak or medium-strength acids.
The subsequent invention describes a process for preparing PNA oligomers of the formula I
in which
R
0
is hydrogen, C
1
-C
18
-alkanoyl, C
1
-C
18
-alkoxycarbonyl, C
3
-C
8
-cycloalkanoyl, C
7
-C
15
-aroyl, C
3
-C
13
-heteroaroyl, or a group which favors intracellular uptake of the oligomer or interacts with the target nucleic acid drawing hybridazation;
A is an amino acid residue, preferably from the group glycine, leucine, histidine, phenylalanine, cysteine, lysine, arginine, aspartic acid, glutamic acid, proline, tetrahydroquinoline-3-carboxylic acid, octahydroindole-2-carboxylic acid and N-(2-aminoethyl)glycine;
k is an integer from 0 to 20, preferably from 0 to 10;
Q is an amino acid residue, preferably from the group glycine, leucine, histidine, phenylalanine, cysteine, lysine, arginine, aspartic acid, glutamic acid, proline, tetrahydroquinoline-3-carboxylic acid, octahydroindole-2-carboxylic acid and N-(2-aminoethyl)glycine;
l is an integer from 0 to 20, preferably from 0 to 10;
B is a nucleotide base which is customary in nucleotide chemistry, for example natural nucleotide bases such as adenine, cytosine, guanine, thymine and uracil, or unnatural nucleotide bases such as purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N
4
,N
4
-ethanocytosine, N
6
,N
6
-ethano-2,6-diaminopurine, 5-methylcytosine, 5-(C
3
-C
6
)-alkynyluracil, 5-(C
3
-C
6
)-alkynylcytosine, 5-fluorouracil or pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyrimidine, or their prodrug forms;
Q
0
is hydroxyl, NH
2
or NHR″, with R″=C
1
-C
18
-alkyl, C
2
-C
18
-aminoalkyl or C
2
-C
18
-hydroxyalkyl; and
n is an integer of 1-50, preferably 4-35,
wherein either amino acids (Q′) are first coupled, using a method which is customary for solid phase synthesis, to a polymeric support of the formula II
L-[polymer]  (II),
which is provided with an anchor group L which contains the radical Q
0
in a latent manner, to result in, as an intermediate, a compound of the formula III
(Q′)
l
-L-[polymer]  (III),
in which L is defined as above, Q′ is an amino acid Q which is, where appropriate, protected in its side chain, and l is an integer from 0 to 20, and
a) a compound of the formula IV
in which
PG is a base-labile amino protecting group, and
B′ is a nucleotide base which is protected on the exocyclic amino function, is then coupled onto the compound of the formula III, or a compound of the formula IV is coupled directly to the polymeric support of the formula II, using the coupling reagents which are customary in peptide chemistry,
b) the temporary, base-labile protecting group PG is eliminated using a suitable reagent,
c) steps a and b are repeated n−1 times,
d) further amino acids A′, which are defined as A but are, where appropriate, protected in their side chains, are coupled on using a method which is customary for solid phase synthesis and then, if R
0
is not hydrogen, the radical R
0
is introduced using a customary method,
e) the compound of the formula I is cleaved, out of the compound of the formula Ia
which is obtained as intermediate compound and in which R
0
, A′, k, B′, n, Q′ and l are defined as above and L is an anchor group, from the polymeric support using a cleaving reagent, with the protecting groups which are, where appropriate, present on the exocyclic amino function of the nucleotide bases and on the side chains of the amino acids being eliminated simultaneously or else subsequently.
Examples of groups which favor the intracellular uptake of the oligomer are alkanoyl and alkoxycarbonyl compounds having various lipophilic radicals such as —(CH
2
)
x
—CH
3
, in which x is an integer of 6-18, —(CH
2
)
n
—CH═CH—(CH
2
)
m
—CH
3
, in which n and m, independently of each other, are an integer from 6 to 12, —(CH
2
CH
2
O)
4
—(CH
2
)
9
—CH
3
, —(CH
2
CH
2
O)
8
—(CH
2
)
13
—CH
3
and —(CH
2
CH
2
O)
7
—(CH
2
)
15
—CH
3
, and also steroid residues, such as cholesteryl, or vitamin residues, such as vitamin E, vitamin A or vitamin D, and other conjugates which make use of natural carrier systems, such as bile acid, folic acid, 2-(N-alkyl, N-alkoxy)-amino-anthraquinone, and conjugates of mannose and peptides of the corresponding receptors which lead to receptor-mediated endocytosis of the oligomers, such as EGF (epidermal growth factor), bradykinin and PDGF (platelet derived growth factor). Labeling groups are understood to mean fluorescent groups, for example of dansyl-(N-dimethyl-1-aminonaphthyl-5-sulfonyl-)derivatives, fluorescein derivatives or coumarin derivatives, or chemiluminescent groups, for example of acridine derivatives, as well as the digoxygenin system, which can be detected using ELISA, the biotin group, which can be detected using the biotin/avidin system, or else linker arms having functional groups which allow subsequent derivatization with detectable reporter groups, for example an aminoalkyl linker which is reacted with an active acridinium easter to form a chemiluminescent probe. Typical labeling groups are:
Groups which upon hybridization of the oligomer with the target nucleic acid attack the latter by binding, cross-linking or clearing are for example conjugates of acridine, psoralene, phenanthridine, naphthoquinone, daunomycin or chloroethylaminoaryl. Typical intercalating and crosslinking residues are:
Anchor groups L, which contain the function Q
0
in a latent manner, are described, for example, in George Barany, Nancy Kneib-Cordonier and Daniel G. Mullen, Int. J. Peptide Protein Res., 1987, 30, 705-739; Gregg B. Fields and Richard L. Noble, Int. J. Peptide Protein Res. 35, 1990, 161-214; K. Barlos, D. Gatos, J. Hondrelis, J. Matsoukas, G. J. Moore, W. Schafer and P. Botiriou, Liebigs Ann. Chem. 1989, 951-955; H. Rink, Tetrahedron Lett. 1987, 3787-3790; G. Breipohl, J. Knoll. and R. Geiger, Tetrahedron Lett. 1987, 5647-5650; G. Breipohl, J. Knolle and W. Stüber, Int. J. Peptide Protein Res., 1989, 34, 262-267; W. Stüber, J. Knolle and G. Breipohl, Int. J. Peptide Protein Res., 1989, 34, 215-220 or in EP-A-0 264 802 (HOE 86/F259), EP-A-0 287 882 (HOE 86/F101) and EP-A-0 322 348 (HOE 87/F386K).
Examples of polymeric supports which are provided with an anchor group, which contains the group Q
0
in a latent manner, are 4-alkoxybenzyl alcohol resin, 2-methoxy-4-alkoxybenzyl alcohol resin, 2-chlorotriphonylmethyl resin, 2,4-dimethoxybenzhydrylamine resin or 4-(2′,4′-dimethoxyphenylaminomethyl)phenoxymethyl resin, or the polymeric supports which are functionalized with a primary a

Affiliated with

Also associated with

Rating

PNA synthesis using a base-labile amino protecting group does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with PNA synthesis using a base-labile amino protecting group, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and PNA synthesis using a base-labile amino protecting group will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2614545