Tehahedron Letters. Vol. 34. No. 36. pp. 5697-5700.1993 00404039193 56.00 + .OO

Printed in Great Britain Pergamon Press Lid

Peptide Analogues of DNA Incorporating Nuckobase-Ala-Pro Subunits

Ian Lewis Sandoz Pharma Ltd., CH4002 Basel, Switzerland

Abstract: The synthesis of building blocks incorporating the nucleobase-Ala-Pro subunit is presented. The synthesis of 1 H*N-(AalaPro)zCralaPro)2(AalaProh_oMe, SDZ 223-586, is described as a representative example.

Interest in the development of peptide analogues of DNA has recently considerably in~reased,~~~~*~~ and prompts us to report the synthesis of 1 HzN-(AalaPro)z(TalaPro)@alaPm~-OMe SDZ 223-586. The most attractive features of this concept include the prospect of making nucleotide analogues that are considerably more stable towards nucleases, and that may exhibit improved transport properties as a result of the uncharged peptide backbone. Compatibility with the flexible and l$hly developed methods of peptide chemistry would also enable synthesis of millimole to mole quantities.

We were convinced that the conformational and thermodynamic properties of the peptide backbone would be decisive in dete rmining the properties of the oligomer. With the desired directionality of pairing in view, a chiral molecular structure for the nucleo amino acid building block was selected. Several spacer amino acids were considered including glycine, &alanine, lysine, proline. hydroxyproline and homoproline. Molecular modelling studies7 were accomplished using the Insight and Discover software packages (Biosym Technologies Inc., San Diego. CA 92121). Structums were optimised using the Discover CVPF force field and the conjugate gradient method. A more detailed presentation of the method and molecular modelling results will be the subject of a forthcoming publication.*

OH B-NH

fR2 -NH

0 L- 3 -8

Nucleo Amino Acid

L- 3 R’=Thymine L- 4 R’=Uracil L- 5 R’=Cytosine L- 6 R1=6-Chloropurine L- 7 R1=2-Arnino-6~chloropmine L- 8 R’=Adenine D- 9 Rt=Adenine

0 D- 9

ProtectirlgGroup

Benxyl

Methoxyethoxymethyl carboxybenxy10xy

The nucleo amino acids 3 - 9 were synthesised in enantiomerically pure form from Boc serine ~_lactone according to Vederas.9*10 Synthesis of the isomeric nucleobases such as iso-cytosine were also investigated by this method. Initial protection studies were carried out with adenine and cytosine using the benxyl group. However it was subsequently decided that the carboxybenxyl group was more ideally suited towards our synthetic proceedures. l1 Guanine was protected as the 06- methoxyethoxymethyl ether, although this was found to be deliterious to Nc regioselectivity. Protection of the guanine 06 with [2-@rhrophenyl)ethyl] may be a route to improved l@ regioselectivity.‘*

The synthesis of oligomers was initiaRy investigated by using solution phase techniques with minimal protection. As a representative example, the synthesis of 1 HzN-(AalaProhCralaProh(AataRo)z-OMe, SDZ 223-586, was carried out. The optimum approach in solution was based on the coupling of two base trimers. Towards this goal, the synthesis of the smino fragment 14 HzN-TalaPro(AalaPro)2-OMe, was accomplished as illustrated in Scheme 1. Standard coupling of 3 Boc-Tala-OH, and proline methyl ester followed by hydraxinolysis and azide coupling with 11 H2N-(AalaPro)~-OMc, gave the base trimer 13

5697

5698

0 0 0

13. R-Boc

14. R=H. H,N-Tak~Pm(AdaPm),Otvle

Scheme 1

Scheme 2 Boc-TaWro(AalaPro)~-OMe. This was subsequently treated with TFA giving the free amino base trimer 14 in 30% yield from 11 as shown in scheme 1. The base dimer 11 H2N-(AalaProh-OMe was prepared analogously.

The Boc protected base trimer 18 Boc-(AalaPro)?TalaPro-OMe was synthesised according to Scheme 2. In contrast to scheme 1. this involved the azide coupling of the base dimer 17 Boc-(AalaProh-Ns with tbe free amino fragment 16 HzN-AalaPro-OMe providing the Boc protected base trimer 18 in 47% yield from 15. Analysis of these oligomers by 4OOMHz ‘H NMR showed no traces of possible diastereomers.

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Final convergent coupling to the target base hexamer 1 was achieved as shown. Conversion of 18 Boc-(AalaPro)~TalaPro-Me to the axide 19 Boc-(AalaPro)2TalaPro-N3 and coupling to the free amino fragment 14 HZN-TalaPro(AalaPro~-Me afforded the base hexamer 20, which was deprotected and purified by reversed phase HPLC to give 1 HzN_(AalaPro)2(TalaProh(AalaPro)2_OMe SDZ 223-586 in 75% yield from 14.

It was considered that mass spectral analysis of model nucleopeptide oligomers by electrospray techniques could lead to novel sequencing methods. This was attempted initially on the model base pentamer 21 HzN-(AalaPro)~alaProh(AalahoXAalaLys)-NH~ , which had been synthesised by solid phase methodology for comparison with solution phase methods. Both the sequence of bases and of spacer amino acids of this oligomer would potentially give rise to fragments valuable for sequencing. Elution of sample was carried out with acetonitrile : water, and the electrospray MS analysis was carried out at orifice tensions of 70,90, 120 and 150 V. The double charged molecular ion (790.4 Da) and the triple charged molecular ion (527.2 Da) were observed defiig a molecular mass of 1578 Da. However, neither fragmentation or signal diminution with loss of sensitivity was observed, indicating molecular ions stable to these conditions. The doubly charged molecular ion (798.4 Da) and the triply charged molecular ion were selected as parent ions for different MS/MS analysis. However, no daughter ions or fragments could be observed.

Nucleopeptide oligomers 20 and 1 were subjected to similar analysis by electrospray at orifice tension of 90 V. Analysis of 20 led to observation of the double charged molecular ion (%2.2 Da) and the triple charged molecular ion (641.6 Da) defining a molecular mass of 1922 Da. In MS/MS analysis the doubly charged molecular ion was observed, and found to be stable.

Analysis of 1 led to observation of the double charged molecular ion (912 Da) and the triple charged molecular ion (608.4 Da) defining a molecular mass of 1821.88 Da. In MS/MS analysis the doubly charged molecular ion (912 Da) and the triply charged molecular ion (608.4 Da) were observed. Although fragmentation of this triply charged ion (698.4 Da) was observed. the intensity was not sufficient for the sequence to be assigned unambiguously.

In conclusion, solution phase methodology was found to be suited to developing strategy for the assembly of oligomers incorporating the nucleobase-Ala-Pro subunit. Analysis of model nucleopeptide oligomers by electrospray MS and MS/MS techniques gave insights into the development of sequencing techniques. Adaption of this approach to solid phase synthesis and biophysical evaluation of the properties of such oligomers encompassing molecular modelling and gel electrophoresis retardation studies will be discussed in a forthcoming publication.

5700

Acknowledgements

The collaboration with Dr. R. Breckenridge in respect of molecular modeling, Dr. H. P. Moser in respect of electrospray M.S./M.S. studies and Dr. D. Cheneval for the gel electrophoresis studies and the major contribution to synthesis of oligomers by Mr. F. Kessler and Mrs. S. Arnold is gratefully acknowledged.

References

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4471, (47), 1982; Ilb. B. E. Watkins, J. S. Kiely and H. Rapoport, J. Am. Chem. Sot. 5702, (104). 1982. 12. T. F. Jenny, S. A. Benner, Tetrahedron Lett. 6619, (33), 1992.

(Received in Germany 4 June 1993; accepted 9 July 1993)