Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Synthesis of peptides
Reexamination Certificate
1999-05-11
2003-05-20
Lukton, David (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Synthesis of peptides
Reexamination Certificate
active
06566494
ABSTRACT:
BACKGROUND OF THE INVENTION
Solid-phase peptide synthesis (SPPS) involves a covalent attachment step (i.e., anchoring) that links the nascent peptide chain to an insoluble polymeric support (i.e., support material) containing appropriate functional groups for attachment. Subsequently, the anchored peptide is extended by a series of addition (deprotection/coupling) cycles that involve adding N
&agr;
-protected and side-chain-protected amino acids stepwise in the C to N direction. Once chain assembly has been accomplished, protecting groups are removed and the peptide is cleaved from the support.
Typically, SPPS begins by using a handle to attach the initial amino acid residue to the functionalized polymeric support. A handle (i.e., linker) is a bifunctional spacer that, on one end, incorporates features of a smoothly cleavable protecting group, and on the other end, a functional group, often a carboxyl group, that can be activated to allow coupling to the functionalized polymeric support. Known handles include acid-labile p-alkoxybenzyl (PAB) handles, photolabile o-nitrobenzyl ester handles, and handles such as those described by Albericio et al.,
J. Org. Chem.,
55, 3730-3743 (1990) and references cited therein, and in U.S. Pat. No. 5,117,009 (Barany) and U.S. Pat. No. 5,196,566 (Barany et al.).
For example, if the support material is prepared with amino-functionalized monomers, typically, the appropriate handles are coupled quantitatively in a single step onto the amino-functionalized supports to provide a general starting point of well-defined structures for peptide chain assembly. The handle protecting group is removed and the C-terminal residue of the N
&agr;
-protected first amino acid is coupled quantitatively to the handle. Once the handle is coupled to the solid-phase and the initial amino acid or peptide is attached to the handle, the general synthesis cycle proceeds. The synthesis cycle generally consists of deprotection of the N
&agr;
-amino group of the amino acid or peptide on the resin, washing, and, if necessary, a neutralization step, followed by reaction with a carboxyl-activated form of the next N
&agr;
-protected amino acid. The cycle is repeated to form the peptide or protein of interest. Solid-phase peptide synthesis methods using functionalized insoluble supports are well known. See, for example, Merrifield,
J. Am. Chem. Soc.,
85, 2149 (1963); Barany and Merrifield, In
The Peptides, Vol.
2, pp. 1-284 (1979); Gross, E. and Meienhofer, J., Eds., Academic: New York; and Barany et al.,
Int. J. Peptide Protein Res.,
30, 705-739 (1987).
Most current methods of SPPS rely on the a-carboxyl function of the eventual C-terminal amino acid residue to achieve anchoring to the support. However, this approach limits SPPS to the formation of peptides having acid, amide, or monosubstituted amide functionality, for example, as the C-terminal functionality, unless more complex procedures are used. Furthermore, certain functionalities, such as aldehydes, cannot typically be obtained using this approach. Cyclic peptides are also not possible using this method. Also, racemization of sensitive amino acid residues in the synthesis of peptide acids is a problem using this method.
Side-chain anchoring, i.e., methods of SPPS that use amino acids with side-chain functional groups for attachment of peptides, is potentially useful for the formation of unusual C-terminal functionalities as well as cyclic peptides. However, side-chain anchoring is inherently limited to certain trifunctional amino acids. Therefore, it would be desirable to develop a general method of SPPS that: (1) allows for the preparation of a wider variety of peptides; (2) does not typically result in racemization of sensitive amino acid residues; and (3) can incorporate a wider variety of amino acids into cyclic peptides.
SUMMARY OF THE INVENTION
The present invention provides a method of forming a support material linked to an amine-containing organic group for solid phase organic synthesis comprising:
(a) attaching a preformed divalent linker to a support material; and
(b) attaching an amine-containing organic group to the preformed divalent linker;
wherein steps (a) and (b) are carried out to form a support material linked to an amine-containing organic group of the following formula (Formula I):
wherein:
(i) Ŝ represents a support material;
(ii) L represents a divalent linker;
(iii) Y represents H or a protecting group; and
(iv) R
1
, R
2
, and R
3
are each independently H or an organic group.
A preferred divalent linker (L) is of the formula (Formula II):
wherein:
each U is independently selected from the group consisting of an alkyl group, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthio group, an arylthio group, an alkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, an alkylsulfoxide group, a halogeno group, and a nitro group, wherein any two U groups can be joined to form a ring; W is a functionalized spacer group for anchoring the linker to the support material; R
5
and R
6
are each independently H, an alkyl group, or an aryl group; and x=0-4.
To synthesize an organic compound, such as a peptide, once the support material of Formula I is prepared, a second organic group is attached to the N atom to build an organic compound. This is done using standard solid phase synthesis techniques and repeated addition cycles of deprotection and coupling.
The present invention also provides a method of synthesizing an organic compound comprising:
(a) providing an aldehyde-functionalized support material having the following formula (Formula III):
wherein:
(i) Ŝ represents a support material;
(ii) V is NH, S or O;
(iii) T is O, NH, NHC(O)R
4
, or S, wherein R
4
is an alkylene group, an arylene group, or an aralkylene group;
(iv) R
7
is an alkylene group, an arylene group, or an oxyalkylene group;
(v) each U is independently selected from the group consisting of an alkyl group, an alkoxy group, an aryl group, an alkoxyaryl group, an aralkyl group, an aralkoxy group, an alkylthio group, an arylthio group, an alkylamido group, an alkylsulfinyl group, an alkylsulfonyl group, an alkylsulfoxide group, a halogeno group, and a nitro group, wherein any two U groups can be joined to form a ring;
(vi) x=0-4; and
(vii) n=1-18;
(b) attaching an amine-containing organic group to the aldehyde functionality under reducing conditions; and
(c) attaching a second organic group to the N atom of the amine-containing organic group to build an organic compound.
The aldehyde-functionalized support material of Formula III is also provided, along with a kit for synthesizing an organic compound. The kit includes an aldehyde-functionalized support material having the Formula III and instructions for preparing an organic compound on the aldehyde-functionalized support material.
The present invention also provides a support material linked to an amine-containing organic group for solid-phase synthesis of an organic compound, wherein the support material has the following formula (Formula I):
wherein:
(a) Ŝ represents a support material;
(b) L represents a divalent linker;
(c) Y represents H or a protecting group;
(d) R
1
and R
2
are each independently H or an organic group; and
(e) R
3
is an organic group having a protecting group Z that is removable under mild conditions.
The present invention also provides a preformed linker having an amine-containing organic group attached thereto, of the formula (Formula IV):
wherein:
(a) L represents a divalent linker;
(b) Q represents a group selected from the group consisting of C(O)OH, C(O)OPfp, C(O)F, C(O)Br, C(O)Cl, OH, Br, Cl;
(c) Y represents H or a protecting group;
(d) R
1
and R
2
are each independently H or an organic group; and
(e) R
3
is an organic group having a protecting group Z that is removable under mild conditions.
As used herein, the term “organic group” means a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of
Albericio Fernando
Alsina Jordi
Barany George
Jensen Knud J.
Songster Micheal F.
Lukton David
Mueting Raasch & Gebhardt, P.A.
Regents of the University of Minnesota
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