Substituted N-ethylglycine derivatives for preparing PNA and...

Details Substituted N-ethylglycine derivatives for preparing PNA and... Substituted N-ethylglycine derivatives for preparing PNA and...

2000-02-18

2002-10-15

Berch, Mark L. (Department: 1624)

Organic compounds -- part of the class 532-570 series

Organic compounds

Pteroyl per se or having -c-, wherein x is chalcogen, bonded...

C544S265000, C544S266000, C544S271000, C544S276000, C544S277000, C544S280000, C544S311000, C544S312000, C544S316000, C544S317000, C548S255000, C548S264400, C548S265400, C548S267200, C548S324100, C548S326500, C548S327100, C548S327500, C548S328500, C548S333100, C548S332500, C548S338100

Reexamination Certificate

active

06465650

ABSTRACT:

Substituted N-ethylglycine derivatives for preparing PNA and PNA/DNA hybrids
The present invention relates to novel substituted N-ethylglycine derivatives for preparing PNA and PNA/DNA hybrids as described in the simultaneously filed application “Peptide oligonucleotide derivatives, their preparation and their use” (HOE 94/F 057, DE-P 44 08 534.6)
Peptide or polyamide nucleic acids (PNA) are DNA-analogous compounds, in which the deoxyribose phosphate backbone was replaced by a peptide oligomer. The syntheses hitherto described in the literature (for example Michael Egholm, Peter E. Nielsen, Rolf H. Berg and Ole Buchardt, Science 1991, 254, 1497-1500; Ole Buchardt, Michael Egholm, Peter E. Nielsen and Rolf H. Berg, WO 92/20702) use, as a temporary protective group for the amino group of the monomer, the acid-labile tert-butyloxycarbonyl (Boc) protective group, which is eliminated by medium-strong acids such as, for example, trifluoroacetic acid. The solid-phase synthesis of oligomers is carried out in accordance with the customary peptide synthesis processes, as they have been described, for example, by Merrifield (B. Merrifield, J. Am. Chem. Soc., 1963, 85, 2149). The PNA oligomer is eliminated with the aid of a strong acid, customarily using liquid hydrogen fluoride.
The repeated treatment with trifluoroacetic acid and the subsequent cleavage using hydrogen fluoride is not compatible with the synthesis of mixed PNA/DNA sequences, since the nucleosidic linkage is not stable under these conditions. In particular, the purine nucleotides deoxyguanosine and deoxyadenosine are rapidly cleaved on the N-glycosidic linkage by strong acids. Moreover, it would be particularly desirable for the synthesis of such molecules to use the customary DNA synthesizers and to largely retain the chemistry used in this apparatus. This also applies to the preparation of PNA sequences with the aid of such apparatus.
It is therefore an aim of the invention to provide glycine derivatives which allow a simple construction of PNA and PNA/DNA hybrids as well as the use of automatic synthesizers.
Substances which are suitable for this purpose are the compounds of the formula I
in which
PG is a urethane-type amino protective group which is labile to weak acids, such as, for example, 1-(1-adamantyl)1-methylethoxycarbonyl (Adpoc), 1-(3,5-di-tert-butylphenyl)1-methylethoxycarbonyl (t-Bumeoc) and 1-methyl-1-(4-biphenyl)ethyloxycarbonyl (Bpoc), 3,5-dimethoxyphenyl-2-propyl-2-oxycarbonyl (Ddz), or a trityl-type amino protective group which is labile to weak acids, such as triphenyl (Trt), (4-methoxyphenyl)diphenylmethyl (Trt), (4-methylphenyl)diphenylmethyl (Mtt), di-(4-methoxyphenyl)phenylmethyl (Dmt) and 9-(9-phenyl)xanthenyl (pixyl),
X is NH, O or S, preferably NH or O,
Y is CH
2
, NH or O, preferably CH
2
, and
B′ are bases customary in nucleotide chemistry, for example natural bases, such as adenine, cytosine, guanine, thymine and uracil, or unnatural 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 and pseudoisocytosine, the exocyclic amino or hydroxyl groups of all of these being protected by suitable known protective groups, such as the benzoyl, isobutanoyl, acetyl, phenoxyacetyl, 4-(t-butyl)benzoyl, 4-(t-butyl)phenoxyacetyl, 4-(methoxy)benzoyl, 2-(4-nitrophenyl)ethyloxycarbonyl, 2-(2,4-dinitrophenyl)ethyloxycarbonyl, 9-fluorenylmethoxycarbonyl, diphenylcarbamoyl or formamidine group, preferably the benzoyl, isobutanoyl, acetyl, phenoxyacetyl, 4-(tbutyl)benzoyl or 4-(methoxy)benzoyl group, and also, in the case of guanine, by a combination of 2-N-acetyl with 6-O-diphenylcarbamoyl, or are base substitute compounds, such as, for example, imidazole, triazole or nitroimidazole, and their salts, preferably their salts with tertiary organic bases, such as, for example, triethylamine or pyridine.
Compounds of the formula I where Y is CH
2
can be obtained, for example, by reacting a compound of the formula II
in which
PG and X are as defined above and,
R
1
is hydrogen or an ester protective group, such as, for example, methyl, ethyl, butyl or 2-(methoxyethoxy)ethyl,
with a compound of the formula III
in which
B′ is as defined above and
Y is CH
2
at 0-45° C., preferably at room temperature, in a suitable solvent, such as, for example, DMF, acetonitrile, dichloromethane or mixtures of these solvents, using a coupling reagent conventionally used in peptide chemistry, such as, for example, carbodiimides, phosphonium reagents, uronium reagents, acid halides or activated esters, to give a compound of the formula IV
in which PG, X, B′ and R
1
are as defined above and subsequently converting this compound to a compound of the formula I by eliminating the ester protective group R
1
under weakly alkaline conditions using alkali metal hydroxide solution, such as, for example, NaOH, LiOH, KOH, or by tertiary amine compounds in water, such as, for example, triethylamine, or else enzymatically with the aid of esterases or lipases at 0-50° C., preferably at room temperature, in a suitable solvent, such as dioxane, water, tetrahydrofuran, methanol, water or mixtures of the solvents.
Activation methods conventionally used in peptide synthesis are described, for example, in Houben-Weyl, Methoden der organischen Chemie [Methods in organic Chemistry], Volume 15/2, Georg Thieme Verlag Stuttgart 1974, or further reagents are described in the particular references, for example BOP (B. Castro, J. R. Dormoy, G. Evin and C. Selve, Tetrahedron Lett. 1975, 1219-1222), PyBOP (J. Coste, D. Le-Nguyen and B. Castro, Tetrahedron Lett. 1990, 205-208), BroP (J. Coste, M.-N. Dufour, A. Pantaloni and B. Castro, Tetrahedron Lett. 1990, 669-672), PyBroP (J. Coste, E. Frerot, P. Jouin and B. Castro, Tetrahedron Lett. 1991, 1967-1970) and uronium reagents, such as, for example, HBTU (V. Dourtoglou, B. Gross, V. Lambropoulou, C. Zioudrou, Synthesis 1984, 572-574), TBTU, TPTU, TSTU, TNTU, (R. Knorr, A. Trzeciak, W. Bannwarth and D. Gillessen, Tetrahedron Letters 1989, 1927-1930), TOTU (EP-A-0 460 446), HATU (L. A. Carpino, J. Am. Chem. Soc. 1993, 115, 4397-4398), HAPyU, TaPipU (A. Ehrlich, S. Rothemund, M. Brudel, M. Beyermann, L. A. Carpino and M. Bienert, Tetrahedron Lett. 1993, 4781-4784), BOI (K. Akaji, N. Kuriyama, T. Kimura, Y. Fujiwara and Y. Kiso, Tetrahedron Lett. 1992, 3177-3180) or acid chlorides or acid fluorides (L. A. Carpino, H. G. Chao, M. Beyermann and M. Bienert, J. Org. Chem., 56(1991), 2635; J.-N. Bertho, A. Loffet, C. Pinel, F. Reuther and G. Sennyey in E. Giralt and D. Andreu (Eds.) Peptides 1990, Escom Science Publishers B. V. 1991, pp. 53-54; J. Green and K. Bradley, Tetrahedron 1993, 4141-4146), 2,4,6-mesitylenesulfonyl-3-nitro-1,2,4-triazolide (MSNT) (B. Blankemeyer-Menge, M. Nimitz and R. Frank, Tetrahedron Lett. 1990, 1701-1704), 2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (TDO) (R. Kirstgen, R. C. Sheppard, W. Steglich, J. Chem. Soc. Chem. Commun. 1987, 1870-1871) or activated esters (D. Hudson) Peptide Res. 1990, 51-55).
Preferred is the use of carbodiimides, for example dicyclohexylcarbodiimide or diisopropylcarbodiimide. Other reagents which are preferably used are phosphonium reagents, such as, for example, PyBOP or PbBroP, uronium reagents, such as, for example HBTU, TBTU, TPTU, TSTU, TNTU, TOTU or HATU, BOI or acid chlorides or acid fluorides.
To synthesize the compounds of the formula II, aminoethylglycine, hydroxyethylglycine, mercaptoethylglycine or their corresponding esters are provided with the corresponding protective group which is labile to weak acids. The protective group which is labile to weak acids is introduced with the aid of processes per se known from the literature, some of which have been modified. Examples of suitable reagents are t-Bumeoc fluoride, Adpoc azide, Bpoc azide, Ddz (phenyl)carbonate, Trt Cl, Mtt Cl, Mmt Cl, Mmt Cl, Dmt Cl, Pixyl Cl. In this reaction, the solubility

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