ALDEHYDE CAPTURE LIGATION TECHNOLOGY FOR SYNTHESIS OF AMIDE BONDS
The present invention relates to ligation agents and their use in making an amide ligation product. Methods of making the ligation agents are also disclosed.
This application is a divisional of U.S. patent application Ser. No. 14/583,586, filed Dec. 26,2014, which claims the benefit of U.S. Provisional Application Ser. No. 61/920,844, filed Dec. 26,2013, which is hereby incorporated by reference in its entirety.
This invention was made with government support under grant number R01 GM073943 awarded by the National Institutes for Health and grant number CHE-1151554 awarded by the National Science Foundation. The government has certain rights in this invention.
FIELD OF THE INVENTIONThis invention is directed to amide ligation agents.
BACKGROUND OF THE INVENTIONMethods for chemoselective formation of amide bonds between unprotected peptide fragments have provided valuable access to recombinant proteins and have become reliable methods for the synthesis of bioconjugates (Muir, Annu. Rev. Biochem. 72:249-289 (2003); Davis, Science 303:480-482 (2004); Nilsson et al., Annu. Rev. Biophys. Biomol. Struct. 34:91-118 (2005); Pattabiraman et al., Nature 480:471-479 (2011); Stephanopoulos et al., Nat Chem Biol 7:876-84 (2011); Hackenberger et al., Angew. Chem. Int. Ed., 47:10030-74 (2008); Ogunkoya et al., Angew. Chem. Int. Ed. 51:9693-97 (2012); Tam et al., Peptide Science 60:194-205 (2001); Mao et al., J. Am. Chem. Soc. 126:2670-71 (2004)). These advances have made total synthesis of therapeutic peptides and proteins, hormones, and modified antibodies a credible objective (Wang et al., Science 342:1357-60 (2013); Payne et al., Chem. Commun. 46:21-43 (2010); Scheck et al., ACS Chem. Biol. 2:247-51 (2007); Gamblin et al., Chem. Rev. 109:131-63 (2008); Chalker et al., Acc. Chem. Res. 44:730-41 (2011); Kiessling et al., Annu. Rev. Biochem. 79:619-53 (2010); Dawson et al., Annu. Rev. Biochem. 69:923-60 (2000); Raibaut et al., Chem. Soc. Rev. 41:7001-15 (2012)). The key concept underlying peptide ligation approaches is that the amide bond formation step can be accelerated by capturing the carboxyl and the amine functionalities and enforcing intramolecular bond formation (
Some shortcomings of these and other methods is that they are not completely general. For example, NCL and most auxiliary-based approaches require the presence of an N-terminal cysteine or modified residues that contain a thiol group (Hackenberger et al., Angew. Chem. Int. Ed. 47:10030-10074 (2008)). Moreover, ligation rate and yield can be inefficient with bulky amino acid residues at the ligation junction (Hackeng et al., Proc. Natl. Acad. Sci. USA 96:10068-10073 (1999)).
The present invention is directed to overcoming these and other deficiencies in the art.
SUMMARY OF THE INVENTIONA first aspect of the present invention relates to a ligation agent of Formula I:
wherein
- A is selected from the group consisting of
fluorescent dyes, and cytotoxic small molecule drugs;
- X is selected from the group consisting of
represents a point of attachment to —C(O)—Se—X—(CR42)mC(O)R3;
represents a point of attachment to —(CR42)mC(O)R3;
represents a point of attachment to —Se—C(O)-A;
- n is 1-3;
- m is 0-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine;
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- R3 is selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when A is (R1)2N(C(R2)2)n— and X is
and with the proviso that m is 0-2 when A is (R1)2N(C(R2)2)n— and X is
A second aspect of the present invention relates to a method of forming an amide ligation product. This method involves reacting a compound containing an amino group with a ligation agent, the ligation agent including a seleno ester group, under conditions effective to produce an amide ligation product.
A third aspect of the present invention relates to a compound of Formula II:
wherein
- X is selected from the group consisting of
- each
represents a point of attachment to —Se—Se—X—(CR42)mC(O)R3;
- each
represents a point of attachment to —(CR42)mC(O)R3;
- each m is independently 0-3;
- each R3 is independently selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when X is
and with the proviso that m is 0-2 when X is
A fourth aspect of the present invention relates to a method of making a ligation agent of Formula I:
wherein
- A is selected from the group consisting of
fluorescent dyes, and cytotoxic small molecule drugs;
- X is selected from the group consisting of
represents a point of attachment to —C(O)—Se—X—(CR42)mC(O)R3;
represents a point of attachment to —(CR42)mC(O)R3;
represents a point of attachment to —Se—C(O)-A;
- n is 1-3;
- m is 0-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine;
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- R3 is selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, C(O)R7, C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when A is (R1)2N(C(R2)2)n— and X is
and with the proviso that m is 0-2 when A is (R1)2N(C(R2)2)n— and X is
- This method includes:
- (i) providing a compound of Formula II:
-
-
- wherein
- X is selected from the group consisting of
-
-
-
- each X is selected from the group consisting of
-
and
-
-
- each
-
represents a point of attachment to —Se—Se—X—(CR42)mC(O)R3;
-
-
- each
-
represents a point of attachment to —(CR42)mC(O)R3;
-
-
- each m is independently 0-3;
- each R3 is independently selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when X is
-
and with the proviso that m is 0-2 when X is
-
- (ii) (a) providing a compound of Formula III:
-
-
- wherein
- A is selected from the group consisting of
-
fluorescent dyes, and cytotoxic small molecule drugs;
represents a point of attachment to —C(O)—OH;
-
-
- n is 1-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine; and
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; or
- (ii) (b) providing a compound of Formula III′:
-
-
-
- wherein
- A is selected from the group consisting of
-
fluorescent dyes, and cytotoxic small molecule drugs;
-
-
- Y is selected from the group consisting of O, S, Se, a good leaving group, and Dawson's auxiliary;
- Y′ is optionally present and, if present, is a C1-C6 alkyl, an ester, or an amide
-
represents a point of attachment to
is a solid support;
-
-
- n is 1-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine; and
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- each R4 is independently selected from the group consisting of H, C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; and
- (iii) reacting the compound of Formula II with the compound of Formula III or the compound of Formula III′ under conditions effective to make a ligation agent of Formula I.
-
Introduced herein is an approach for amide ligation, i.e., aldehyde capture ligation (“ACL”), that is, in principle, applicable to any N-terminal amino acid residue for peptide synthesis while allowing rapid ligation of challenging residues. The method is general, beyond peptide synthesis, for the formation of an amide bond between any carboxylic acid and amine. One feature of ACL is that it employs the rapid association between an aldehyde group and an amine to enforce an intramolecular reaction leading to the desired native amide bond formation. ACL also allows for the synthesis of large molecules.
One aspect of the present invention relates to a ligation agent of Formula I:
wherein
- A is selected from the group consisting of
fluorescent dyes, and cytotoxic small molecule drugs;
- X is selected from the group consisting of
represents a point of attachment to —C(O)—Se—X—(CR42)mC(O)R3;
represents a point of attachment to —(CR42)mC(O)R3;
represents a point of attachment to —Se—C(O)-A;
- n is 1-3;
- m is 0-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine;
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- R3 is selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when A is (R1)2N(C(R2)2)n— and X is
and with the proviso that m is 0-2 when A is (R1)2N(C(R2)2)n— and X is
As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings. If not defined otherwise herein, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.
As used herein, the term “alkyl” means an aliphatic hydrocarbon group which may be straight or branched having about 1 to about 6 carbon atoms in the chain. Branched means that one or more lower alkyl groups such as methyl, ethyl, or propyl are attached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, and 3-pentyl.
The term “alkenyl” means an aliphatic hydrocarbon group containing a carboncarbon double bond and which may be straight or branched having about 2 to about 6 carbon atoms in the chain. Preferred alkenyl groups have 2 to about 4 carbon atoms in the chain. Exemplary alkenyl groups include ethenyl, propenyl, n-butenyl, and i-butenyl.
The term “alkynyl” means an aliphatic hydrocarbon group containing a carboncarbon triple bond and which may be straight or branched having about 2 to about 6 carbon atoms in the chain. Preferred alkynyl groups have 2 to about 4 carbon atoms in the chain. Exemplary alkynyl groups include ethynyl, propynyl, n-butynyl, 2-butynyl, 3-methylbutynyl, and n-pentynyl.
As used herein, the term “aryl” refers to an aromatic monocyclic or polycyclic ring system containing from 6 to 19 carbon atoms, where the ring system may be optionally substituted. Aryl groups of the present invention include, but are not limited to, groups such as phenyl, naphthyl, azulenyl, phenanthrenyl, anthracenyl, fluorenyl, pyrenyl, triphenylenyl, chrysenyl, and naphthacenyl.
The term “alkoxy” means groups of from 1 to 8 carbon atoms of a straight, branched, or cyclic configuration and combinations thereof attached to the parent structure through an oxygen. Examples include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like. Lower-alkoxy refers to groups containing one to four carbons. For the purposes of the present patent application, alkoxy also includes methylenedioxy and ethylenedioxy in which each oxygen atom is bonded to the atom, chain, or ring from which the methylenedioxy or ethylenedioxy group is pendant so as to form a ring. Thus, for example, phenyl substituted by alkoxy may be, for example,
The term “halogen” means fluorine, chlorine, bromine, or iodine.
An amino acid according to this and all aspects of the present invention can be any natural or non-natural amino acid.
A “peptide” as used herein is any oligomer of two or more natural or non-natural amino acids, including alpha amino acids, beta amino acids, gamma amino acids, L-amino acids, D-amino acids, and combinations thereof In preferred embodiments, the peptide is ˜5 to ˜30 (e.g., ˜5 to ˜10, ˜5 to ˜17, ˜10 to ˜17, ˜10 to ˜30, or ˜18 to ˜30) amino acids in length. Typically, the peptide is 5-17 amino acids in length.
Amino acid side chains according to this and all aspects of the present invention can be any amino acid side chain from natural or nonnatural amino acids, including from alpha amino acids, beta amino acids, gamma amino acids, L-amino acids, and D-amino acids.
Protecting groups function primarily to protect or mask the reactivity of functional groups. Protecting groups that are suitable for the protection of an amine group are well known in the art, including without limitation, carbamates, amides, N-alkyl and N-aryl amines, imine derivatives, enamine derivatives, and N-hetero atom derivatives as described by THEODORA W. GREENE & PETER G. M. WUTS, P
In some preferred embodiments X in Formula I is
Suitable ligation agents according to this embodiment include, e.g.,
wherein R3 is an aryl or an alkyl;
wherein R3 is an aryl or an alkyl.
In some preferred embodiments X in Formula I is
Suitable ligation agents according to this embodiment include, e.g.,
wherein R3 is an aryl or an alkyl.
A second aspect of the present invention relates to a method of forming an amide ligation product. This method involves reacting a compound containing an amino group with a ligation agent that includes a seleno ester group, under conditions effective to produce an amide ligation product.
Suitable amide ligation products that can be formed using the method of this aspect of the present invention include those having the formula A-C(O)—N(R9)—B, wherein
- A and B are each independently selected from the group consisting of H, (R1)2N(C(R2)2)n—, —NR5R6, C1C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl,—OH, C1-C6 alkoxy, aryl, amino acids, peptides, proteins, carbohydrates, nucleic acids, cytotoxic small molecule drugs, dyes, and polymers;
- n is 1-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine;
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; and
- R9 is selected from the group consisting of H, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, amino acids, peptides, proteins, carbohydrates, and nucleic acids.
In this and all aspects of the present invention, suitable cytotoxic small molecule drugs include, without limitation, cisplatin, doxorubicin, paclitaxel, camptothecin, and doxorubicin.
In this and all aspects of the present invention, suitable dyes include, for example, fluorescein, rhodamine, diazobenzene, BODIPY, and ALEXA.
In this and all aspects of the present invention, suitable polymers include, without limitation, N-(2-hydroxypropyl) methacrylamide (HPMA), poly(ethylene glycol) (PEG), poly(lactide-co-glycolide) (PLGA), PAMAM dendrimers, poly(L-lysine), poly(L-glutamic acid), poly ((N-hydroxyalkyl)glutamine), dextrins, hydroxyethylstarch (HES), polysialic acid, the polyacetal Fleximer, poly-(acrylamide) (PAAm), poly(methacrylic acid) (PMAA), poly-(acrylic acid) (PAA), poly(2-(dimethylamino)ethyl meth-acrylate (PDMAEMA), and poly(N-isopropylacrylamide) (poly(NIPAAM). In at least one embodiment, the polymer is PEG.
Suitable compounds containing an amino group that can be used in this aspect of the present invention include, without limitation, amino acids, peptides, proteins, carbohydrates, nucleic acids, and compounds having the formula B—N(R9)H, wherein
- B is selected from the group consisting of H, (R1)2N(C(R2)2)n—, —NR5R6, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl,—OH, C1-C6 alkoxy, aryl, amino acids, peptides, proteins, carbohydrates, nucleic acids, cytotoxic small molecule drugs, dyes, and polymers;
- R5 and R6 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; and
- R9 is selected from the group consisting of H, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, amino acids, peptides, proteins, carbohydrates, and nucleic acids.
Suitable ligation agents that can be used in this aspect of the present invention include those having the formula:
wherein
- A is selected from the group consisting of H, (R1)2N(C(R2)2)n—, —NR5R6, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl,—OH, C1-C6 alkoxy, aryl, amino acids, peptides, proteins, carbohydrates, nucleic acids, cytotoxic small molecule drugs, dyes, and polymers;
- X is selected from the group consisting of
represents a point of attachment to —C(O)—Se—X—(CR42)mC(O)R3;
represents a point of attachment to —(CR42)mC(O)R3;
represents a point of attachment to —Se—C(O)-A;
- n is 1-3;
- m is 0-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine;
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- R3 is selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when A is (R1)2N(C(R2)2)n— and X is
and with the proviso that m is 0-2 when A is (R1)2N(C(R2)2)n— and X is
In some preferred embodiments, the ligation agent is a compound of Formula I.
A third aspect of the present invention relates to a compound of Formula II:
wherein
- each X is selected from the group consisting of
- each
represents a point of attachment to —Se—Se—X—(CR42)mC(O)R3;
- each
represents a point of attachment to —(CR42)mC(O)R3;
- each m is independently 0-3;
- each R3 is independently selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when X is
and with the proviso that m is 0-2 when X is
In some embodiments X in Formula II is
Suitable compounds according to this embodiment include, e.g.,
wherein R3 is an aryl or an alkyl;
wherein R3 is an aryl or an alkyl.
In some embodiments X in Formula II is
Suitable compounds according to this embodiment include, e.g.,
wherein R3 is an aryl or an alkyl.
A fourth aspect of the present invention relates to a method of making a ligation agent of Formula I. This method involves:
- (i) providing a compound of Formula II;
- (ii) (a) providing a compound of Formula III:
-
- wherein
- A is selected from the group consisting of
- wherein
fluorescent dyes, and cytotoxic small molecule drugs;
represents a point of attachment to —C(O)—OH;
-
-
- n is 1-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine; and
- each R2 is independently selected from the group consisting of H, C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; or
-
- (ii) (b) providing a compound of Formula III′:
-
- wherein
- A is selected from the group consisting of
fluorescent dyes, and cytotoxic small molecule drugs;
-
- Y is selected from the group consisting of O, S, Se, a good leaving group, and Dawson's auxiliary;
- Y′ is optionally present and, if present, is a C1-C6 alkyl, an ester (e.g., —C(O)—O—), or an amide (e.g., —N(R)—, where R is H, C1-C6 alkyl, or aryl);
represents a point of attachment to
is a solid support;
-
- n is 1-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine; and
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; and
- reacting the compound of Formula II with the compound of Formula III or the compound of Formula III′ under conditions effective to make a ligation agent of Formula I.
As will be apparent to the skilled artisan, compounds of Formula II can be prepared by providing a substituted halogenated benzene aldehyde or substituted halogenated benzene ketone and reacting the substituted halogenated benzene aldehyde or substituted halogenated benzene ketone with a diselenide in the presence of a cation scavenger under conditions effective to produce a compound of Formula II.
Suitable solid supports for use in compounds of Formula III′ include, for example, Tenta gel, Rink amide resin, Merrifield resin, trityl resin, PAM resin, and Kaiser resin.
In at least some embodiments, the compound of Formula III′ contains a good leaving group. Leaving groups are displaced as stable species taking with it the bonding electrons, resulting in coupling of one compound to another. Good leaving groups that are suitable in the methods of the present invention are well known in the art and include, without limitation, those employed in standard solution or solid phase peptide synthesis.
Suitable substituted halogenated benzene aldehydes and substituted halogenated benzene ketones include those of Formula IV:
Suitable diselenides include, without limitation, any alkaline metal diselenide, such as sodium diselenide, potassium diselenide, lithium diselenide, etc.
Suitable cation scavengers will be apparent to the skilled artisan. Exemplary cation scavengers include, without limitation, HMPA, DMPU, DMI, tetraalkyl ureas, and cyclic alkyl ureas.
Described herein are compounds and methods useful for, e.g., aldehyde capture ligation (“ACL”) (Kemp, Biopolymers 20:1793-804 (1981); Kemp et al., J. Org. Chem. 40:3003-3004 (1975), each of which is hereby incorporated by reference in its entirety), which utilizes an o-selenobenzaldehyde ester to enforce an intramolecular reaction between the carboxyl and the amine partners (Coltart, Tetrahedron 56:3449-91 (2000), which is hereby incorporated by reference in its entirety). As shown generally in
The 1,3-O→N acyl transfer was postulated based on suggestions of a similar rearrangement underlying the amide bond forming reaction between carboxylic acids and isonitriles (Li et al., J. Am. Chem. Soc. 130:13225-27 (2008), which is hereby incorporated by reference in its entirety). The ortho-aldehyde group in the auxiliary was designed to reversibly capture the amine and intramolecularly deliver it to an activated acid. The selenoesters may also directly condense with the amine (Durek & Alewood, Angew. Chem. Int'l Ed. 50:12042-45 (2011); McGrath & Raines, Acc. Chem. Res. 44:752-61 (2011); Mautner et al., J. Am. Chem. Soc. 85:3458-62 (1963); Chu & Mautner, J. Org. Chem. 31:308-12 (1966), each of which is hereby incorporated by reference in its entirety), although studies described herein suggest that this is not a major pathway. Prior efforts of Kemp et al. investigated auxiliaries derived from naphthyl oxo-esters and observed the formation of the competing imine byproduct which did not convert to the amide (Kemp et al., J. Org. Chem. 40:3003-04 (1975), which is hereby incorporated by reference in its entirety). The present invention uses a more reactive selenoester, and a less rigid scaffold, to favor the acyl transfer.
The present invention may be further illustrated by reference to the following examples.
EXAMPLES Example 1 General Materials and MethodsAll commercial materials (Aldrich, Fluka, Nova) were used without further purification. All solvents were reagent grade or HPLC grade (Fisher). Anhydrous THF, diethyl ether, CH2Cl2, and DMF were obtained from a dry solvent system (passed through column of alumina) and used without further drying. All reactions were performed under an atmosphere of pre-purified anhydrous argon or nitrogen. Yields refer to chromatographically pure compounds; % conversions were obtained by comparison of HPLC peak areas of products and starting esters. Thin Layer Chromatography or HPLC was used to monitor reaction progress. Flash column chromatography was performed using Silica Gel 60 Å (32-63 micron).
Example 2 Nuclear Magnetic ResonanceProton NMR spectra were recorded on a 400 MHz spectrometer and carbon NMR spectra on a 101 MHz, spectrometer at ambient temperature. All NMR chemical shifts (δ) are referenced in ppm relative to residual solvent or internal tetramethylsilane. Solvent reference ppm in 1H-NMR and 13C-NMR for CDCl3 are 7.26 ppm and 77.16 ppm, respectively. Carbon NMR spectra are proton decoupled. NMR spectral data are reported as chemical shift (multiplicity, coupling constants (J), integration). Multiplicity is reported as follows: singlet (s), broad singlet (bs), doublet (d), doublet of doubles (dd), doublet of triplet (td), triplet (t), and multiplet (m). Coupling constant (J) in Hertz (Hz).
Example 3 High-Performance Liquid ChromatographyPreparative HPLC chromatography (HPLC) was performed on Beckman Coulter equipped with System Gold 168 detector and 125P solvent module HPLC with a 10 mm C-18 reversed-phase column. All separations involved a mobile phase of 0.1% TFA (v/v) in water (solvent A) and 0.1% TFA (v/v) in acetonitrile (solvent B).
Analytical HPLC chromatography (HPLC) was performed on an Agilent 1200 series HPLC equipped with a 4.6 mm C-18 reversed-phase column. All separations involved mobile phase of 0.1% TFA (v/v) in water (solvent A) and 0.1% TFA (v/v) in acetonitrile (solvent B). The detection wavelength was set to 263 and 280 nm. Products were not calibrated by internal standard unless otherwise specified. % Conversion was calculated as the integrated area of the peptide product divided by the sum of the areas of the starting material and peptide product.
For determination of reaction rates, the reaction mixture was diluted 20-fold in acetonitrile and kept over dry-ice until HPLC analysis. HPLC (C-18 columns): 0.1% TFA (v/v) in water (solvent A): acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min, detection wavelength 280 nm.
Example 4 FT-IR SpectroscopyAttenuated total reflectance-infrared (ATR-IR) spectra were obtained on the Thermo Nicolet AVATAR FTIR Spectrometer; ν (cm−1) are partially reported.
Example 5 Thin Layer ChromatographyAnalytical thin-layer chromatography (TLC) was performed using Silica Gel 60 Å F254 pre-coated plates (0.25 mm thickness) and visualized using irradiation by a UV lamp and/or staining with I2/silica.
Example 6 Mass SpectrometryMass spectrometry was performed using ultra high performance liquid chromatography-mass spectrometry using the Agilent 1100 Series LCMSD VL MS Spectrometer. High-resolution liquid chromatography-mass spectrometry (HRMS) was performed on an Agilent 6224 TOF LC/MS Mass Spectrometer. Protein MALDI data was collected on Bruker MALDI-TOF/TOF UltrafleXtreme MS Spectrometer. MS/MS analysis was performed on Agilent 1100 Series LCMSD VL MS Spectrometer.
Example 7 Fmoc Solid-Phase Peptide SynthesisAutomated peptide synthesis was performed on CEM Liberty microwave peptide synthesizer. Peptides were synthesized using Fmoc solid-phase chemistry (F
To a 100-mL oven dried two neck round-bottom flask containing PCC (2.33 g, 10.57 mmol) in 10 mL anhydrous dichloromethane (CH2Cl2) was added a solution of 2-mercaptobenzyl alcohol (0.61 g, 4.30 mmol) in anhydrous CH2Cl2 (6 mL) in a dropwise fashion. The mixture was stirred at room temperature for 5 hours and diluted with CH2Cl2 (2×10 mL). The diluted mixture was washed with H2O (2×20 mL). The CH2Cl2 layer was passed through a short pad of Florisil, and concentrated under vacuum to afford SI1 (also referred to as 6 herein) as a white solid; yield: 0.30 g (40%) (Kasmai et al., Synthesis 1989:763-65 (1989), which is hereby incorporated by reference in its entirety). TLC(EtOAc:Hexane 1:3), Rf=0.3, irradiated by a UV lamp. 1H NMR (
A solution of Na2S.9H2O (864.7 mg, 3.6 mmol) in N,N-dimethylacetamide (DMAc, 10 mL) was heated to 80° C. under N2. To this solution, 4-chlorobenzaldhyde (420 mg, 3 mmol) was added and solution was stirred at 80° C. for 1 hour. The resulting mixture was cooled in ice bath, and benzoyl chloride (673 mg, 4.5 mmol) was added dropwise, and the reaction mixture was stirred at 0° C. for another 1 hour. It was then poured into water (30 mL) and extracted with diethyl ether (20 mL×3). The organic layers were combined, washed with saturated NaHCO3 solution, brine and dried over MgSO4. The solution was filtered, concentrated, and purified by flash chromatography (20% ethyl acetate in hexane) to afford SI1A as a white solid; yield 401 mg, 55%. TLC (EtOAc:Hexane 1:4), Rf=0.4, irradiated by a UV lamp. 1H NMR (
Thiosalicylic acid (6.0 g, 39 mmol), LiH (0.80 g, 100 mmol) and anhydrous THF (25 mL) were added to an oven dried round bottom flask. The resulting solution was refluxed under N2 for 1 hour. The resulting mixture was cooled to room temperature, and 50 mL of 1.8 M PhLi in Et2O was added slowly and the mixture stirred at 22° C. After 14 hours, 50 mL of H2O was added to the reaction mixture and the organic layer was separated, concentrated, and purified by flash chromatography to obtain SI2 as a light yellow solid; yield: 2.66 g (32%) (Devarie-Baez et al., Org. Lett. 12:752-754 (2010), which is hereby incorporated by reference in its entirety). TLC (EtOAc:Hexane, 1:3), Rf=0.5, irradiated by a UV lamp. 1H NMR (
Na (0.46 g, 20 mmol) and a catalytic amount of naphthalene (˜1 mmol) were added in anhydrous THF under N2 in an oven dried round bottom flask. The suspension was stirred at room temperature for 1 hour, during which time it turned deep blue. Se (2.0 g, 25 mmol) was added and the solution was refluxed under N2 for 12 hours. The mixture was cooled to room temperature, 2-bromobenzaldehyde (3.3 g, 18 mmol) and DMPU (6 mL) were added, and the resulting mixture was refluxed for 20 hours. After cooling, the reaction mixture was poured into water (50 mL) and extracted with CH2Cl2 (3×20 mL). The combined organic layers were washed with water and dried over Na2SO4. The solvent was concentrated under vacuo and the residue was purified by flash chromatography to obtain SI3 (also referred to as 7 herein) as a yellow solid; yield 1.03 g (28%) (Syper et al., Tetrahedron 44:6119-6130 (1988), which is hereby incorporated by reference in its entirety). TLC (EtOAc:Hexane 1:3), Rf=0.3, irradiated by a UV lamp. 1H NMR (
Amino acid (AA)/peptide (1 mmol) was dissolved in anhydrous THF (5 mL) in an oven-dried round bottom flask under N2. The solution was stirred and cooled at 0° C. in an ice-bath for 10 minutes. Then, DCC (1 mmol) and DMAP (0.25 mmol) were added at 0° C. The reaction was further stirred at 0° C. for 30 minutes. Then, salicylaldehyde or thio benzophenone (1 mmol) was added. The resulting mixture was stirred at room temperature for 2 hours. The progress of the reaction was analyzed by TLC after regular intervals of time. After the completion of the reaction, it was cooled in an ice bath and filtered under vacuum to remove dicyclohexyl urea. The filtrate was concentrated under vacuum and the residue was purified by flash chromatography to give AA/peptide-salicylaldehyde ester or AA/peptide-thio-benzophenone ester as a white solid; yield 60-85%.
Example 13 General Procedure for Synthesis of Thiobenzaldehyde and Selenobenzaldehyde Derived EstersAmino acid (AA)/peptide (1 mmol) was dissolved in anhydrous THF (5 mL) in an oven-dried round bottom flask under N2. The resulting solution was stirred and cooled at 0° C. in an ice-bath for 10 minutes. Then, DCC (1 mmol) was added at 0° C. The reaction was stirred at 0° C. for 30 minutes. Then, di-thio (SI1) or di-seleno benzaldehyde (S13) (0.5 mmol), TCEP.HCl (0.6 mmol), Et3N (0.6 mmol), and 2 drops of water were added. The resulting reaction mixture was stirred at room temperature for 30 minutes. The reaction was then cooled in ice bath and filtered to remove dicyclohexyl urea. The filtrate was concentrated under vacuum and purified by flash chromatography or HPLC to give AA/peptide-thio-benzaldehyde or AA/peptide-seleno-benzaldehyde ester, respectively, as a pale yellow solid (55-70% yield).
Example 14 General Procedure for the Solid Phase Synthesis of Peptide Seleno-EstersThe peptide N-acyl-benzimidazolinone (Nbz) was prepared according to procedure described by Blanco-Canosa & Dawson, Angew. Chem. Int. Ed., 47:6851-55 (2008), which is hereby incorporated by reference in its entirety). The PAM-resin bound Nbz peptide was treated with TFA:TIPS:anisole:thioanisole (95:2.5:2.5:1) for 1 hour to remove the side-chain protecting groups. The on-resin selenolysis for the synthesis of peptide seleno-ester was conducted by adding diselenide (2.0 eq) in Nbz-peptide (1.0 eq) in a dried round bottom flask, thoroughly flushed with argon. Then, dry THF (0.5 mL) and tributylphosphine (2.0 equiv.), flushed with argon, were added to the round bottom flask. The resulting suspension was stirred on heated shaker for 14 hours at 55° C. The resin was then filtered and washed with THF/DCM. For complete removal of peptide from the resin, selenolysis was repeated twice on the same resin. The combined filtrates were concentrated and the crude product was triturated with cold diethyl ether to give a white suspension, which was centrifuged and the ether subsequently decanted to obtain crude peptide-selenoester in 59% yield from the resin. The yield of the peptide was determined using Fmoc absorbance.
Example 15 Synthesis of Salicylaldehyde Ester SI4Salicylaldehyde ester SI4 was synthesized as described in Example 12. Isolated yield: 82%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=24.2 min. 1H NMR (
Oxo-ester SI5 was synthesized as described in Example 12. Isolated yield: 85%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=19.9 min. 1H NMR (
Thio-benzaldehyde ester SI6 was synthesized as described in Example 13. Isolated yield: 60%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=20.9 min. 1H NMR (
Thio-phenyl ester SI7 was synthesized as described in Example 12. Isolated yield: 83%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=22.4 min. 1H NMR (
Thio-benzophenone ester SI8 was synthesized as described in Example 12. Isolated yield: 72%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A): acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=25.1 min. 1H NMR (
Seleno-benzaldehyde ester SI9 was synthesized as described in Example 13. Isolated yield: 69%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A): acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=21.5 min. 1H NMR (
Seleno-phenyl ester SI10 was synthesized as described in Example 13. Isolated yield: 75%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=23.4 min. 1H NMR (
Seleno-benzaldehyde ester SI11 (also referred to as 3 herein) was synthesized as described in Example 13 (Li et al., Org. Lett. 12:1724-27 (2010), which is hereby incorporated by reference in its entirety). Isolated yield: 66%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=22.8 min. 1H NMR (
Seleno-phenyl ester SI12 (also referred to as 4 herein) was synthesized as described in Example 13 (F
Seleno-benzaldehyde ester SI13 was synthesized as described in Example 13. Isolated yield: 58%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=26.4 min. 1H NMR (
Seleno-phenyl ester SI14 was synthesized as described in Example 13. Isolated yield: 68%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=27.1 min. 1H NMR (
Seleno-benzaldehyde ester SI15 was synthesized as described in Example 13. Isolated yield: 63%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=27.2 min. 1H NMR (
Seleno-benzaldehyde ester SI16 was synthesized as described in Example 13. Isolated yield: 70%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A): acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=25.0 min. 1H NMR (
Seleno-benzaldehyde ester SI17 was synthesized as described in Example 13. Isolated yield: 55%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=23.2 min. 1H NMR (
Seleno-benzaldehyde ester SI18 was synthesized as described in Example 13. Isolated yield: 67%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=26.1 min. 1H NMR (
Fmoc-proline selenophenylester SI18A was synthesized as described in Example 13. Isolated yield: 66%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A): acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=23.4 min. 1H NMR (
Seleno-benzaldehyde ester SI19 was synthesized as described in Example 13. Isolated yield: 56%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=21.9 min. 1H NMR (
Seleno-benzaldehyde ester SI20 was synthesized as described in Example 13. Isolated yield: 59%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=26.1 min. 1H NMR (
Seleno-benzaldehyde ester SI20A was synthesized as described in Example 13. Isolated yield: 58%. TLC (EtOAc:Hexane 3:7), Rf=0.4, irradiated by a UV lamp. 1H NMR (
Fmoc-Valine (1.02 g, 3 mmol) and N-hydroxysuccinimide (0.345 g, 3 mmol) were dissolved in dimethoxy ethane (35 ml) and cooled in an ice bath, then DCC (0.681 g, 3.3 mmol) was added. The resulting mixture was stirred in the ice bath for 3 hours, then at room temperature for 20 hours. The precipitate formed was filtered off and the filtrate concentrated under vacuo. The crude product was further purified by flash chromatography (ethyl acetate/hexane, v:v, 4:6) to afford SI20B as a white solid. Isolated yield: 65%. TLC (EtOAc:Hexane 3:2). Rf=0.57, irradiated by a UV lamp. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min. Retention time (Rt)=15.77 min. 1H NMR (
4-(Phenylazo)benzoic acid (67.8 mg, 0.3 mmol), DCC (61.8 mg, 0.3 mmol), and di-seleno benzaldehyde (55.1 mg, 0.15 mmol) in a 10 ml round bottom flask was flushed with nitrogen for 20 minutes. Dry THF (3 ml) was added and the mixture was stirred under nitrogen for 40 minutes. A solution of n-butyl phosphine (0.4 mmol) in 1.5 ml THF and 40 μl H2O was added into the reaction mixture. The resulting mixture was stirred for another 1.5 hour, filtered, concentrated under vacuo, and purified by silica gel chromatography (15% ethyl acetate in hexane). The product obtained from chromatography still contained some impurities, which could be further purified by washing with hexane to afford pure 10 as a red solid (70.1 mg, 60%). Isolated yield: 60%. TLC (EtOAc:Hexane 1:5), Rf=0.57, irradiated by a UV lamp. 1H NMR (
To a solution of C-terminal amino acid or peptide-oxo, thio, or seleno ester (2-10 μmol) in 1 mL DMF, was added N-terminal amino acid or peptide (20 μmol) and Et3N (20 μmol). The reaction was stirred at 22° C. and monitored by analytical HPLC at regular intervals. The reaction mixture was lyophilized, or concentrated under vacuum, and purified by HPLC. HPLC: 0.1% TFA (v/v) in water (solvent A):acetonitrile (solvent B); gradient 45-85% in 30 min, flow rate=0.5 mL/min, detection wavelength 280 nm. This procedure was used to synthesize the pepides shown in Table 1 below.
Two sets of tetrapeptides were prepared and tested to evaluate the extent of epimerization of the selenobenzaldehyde ester: (i) FmocVal-Ala-L-Ala-Gln-NH2 and its expected epimerization product, FmocVal-Ala-D-Ala-Gln-NH2, and (ii) FmocAla-Val-L-Ala-Gln-NH2 and its expected epimerization product, FmocAla-Val-D-Ala-Gln-NH2. The reactive selenobenzaldehyde esters in (i) and (ii) consist of alanine and valine residues, respectively. Rates of condensation of alanine selenobenzaldehyde ester is faster than the valine derivative. Comparison of the two sets of amino acid selenobenzaldehyde esters allows for the critical evaluation of the extent of racemization.
Example 33 Ubiquitin LabelingA solution of Azodye-selenoester (500 μmol) in 10 μl DMF was mixed with ubiquitin (15N labeled) (100 μmol) predissolved in 90 μl 1× phosphate buffered saline (PBS), pH 7.0. The reaction mixture was shaken and the progress monitored by MALDI-TOF Mass Spectrometry. See
As shown in Table 2 below, ACL auxiliary development studies were begun by condensing salicylaldehyde-derived benzylester 1a with benzylamine in DMF (Kemp D. S., Biopolymers 20:1793-804 (1981); Kemp et al., J. Org. Chem. 40:3003-04 (1975), each of which is hereby incorporated by reference in its entirety). Complete conversion of the ester to the amide 2 was observed in less than 5 hours as monitored by LCMS. Presence of the ortho-aldehyde group is critical for the ACL mechanism as phenyl and para-hydroxybenzaldehyde esters, 1b and 1c, respectively react at much slower rates. Compound 1c also served as a control for evaluating potential reactivity increases due to the presence of an electron-withdrawing group at the ortho or para positions of the aromatic ring. Aminolysis reactions are not considered to be sensitive to minor pKa differences in the leaving phenolate ions, as the formation of the tetrahedral transition state is the rate limiting step; although, analysis is complicated by proton transfers (Holmquist et al., J. Am. Chem. Soc. 91:2985-93 (1969); Jencks et al., J. Am. Chem. Soc. 90:2622-37 (1968); Trmcic et al., Beilstein J. Org. Chem. 6:732-41 (2010); Yang et al., Org. Lett. 2:4133-36 (2000), each of which is hereby incorporated by reference in its entirety). The pKa's of the ortho-formylphenol is slightly lower than the para substituted analog while both pKa's are much lower than that of the basic amine nucleophile. The para-benzaldehyde ester analog, 1c, undergoes amide bond formation much slower than the ortho analog 1a reflecting the faster rate of reaction with the aldehyde group than direct acylation of the substituted phenylesters. While the transient hemiaminal/imine in the ortho analog is rapidly captured for acylation, a similar step is not possible for the para analog. Although direct acylation of amines cannot be ruled out as a minor contributor to the product formation, these studies support the aldehyde capture mechanism.
To further enhance the efficiency of the ACL reaction, the leaving group was converted from a phenol moiety to a thiophenol group. The importance of an ortho-aldehyde group for ACL is also evident in the thioester series 1d-1f, as 1d, with an ortho-aldehyde group, reacts more efficiently than the unsubstituted thiophenyl and para-thiobenzaldehyde esters, 1e and 1f, respectively (Table 2). Extensive analysis of aminolysis of thioesters suggests that N-acylations also proceed through tetrahedral intermediates as oxoesters (Yang et al., Org. Lett. 2:4133-36 (2000); Castro, Chem. Rev. 99:3505-24 (1999), each of which is hereby incorporated by reference in its entirety). (The thiobenzaldehyde ester derivatives undergo a side reaction; this reaction can be suppressed by converting the benzaldehyde to the corresponding benzophenone (see
To further accelerate the ACL reaction, the leaving group was converted from a thiophenol moiety to selenophenol derivative 1g (Table 2) (Durek et al., Angew. Chem. Int. Ed. 50:12042-45 (2011); Mautner et al., J. Am. Chem. Soc., 85:3458-62 (1963); Chu et al., J. Org. Chem. 31:308-12 (1966), each of which is hereby incorporated by reference in its entirety). As predicted, substitution with selenol provided a significant boost to the reaction rate with complete conversion to amide 2 occurring in less than one minute under the reaction conditions. Amino acid derived selenobenzaldehyde esters also provide the desired dipeptides more efficiently than the oxo- and thiobenzaldehyde ester analogs (see
The above results support the aldehyde capture and acyl transfer mechanism outlined for aldehyde capture ligation, although the exact identity of the intermediate remains to be probed. The ACL reaction between alanine-derived selenobenzaldehyde ester, 3, and tryptophan methyl ester follows a second order rate constant (FmocAla-COSe-o-PhCHO, k=1.65±0.06 M−1S−1) (Table 3 below and
The compatibility of ACL for synthesis of peptides was determined in aqueous buffers (with DMF as a co-solvent) for potential applications aimed at synthesis of large peptides and proteins or other bioconjugates by fragment coupling. The formation and reactions of hemiaminals and imines are known to be sensitive to buffer pHs (W
In the exploratory studies designed to determine the scope of ACL, the amino acid and peptide thio- and seleno-benzaldehyde esters were initially prepared by coupling activated carboxylic acids and o-benzaldehyde disulfide (6) or diselenide (7) in the presence of a reducing agent, such as TCEP (
Epimerization of activated amino acids is a critical concern in peptide synthesis. The selenobenzaldehyde bearing peptide may be expected to undergo epimerization during the course of aldehyde capture ligation, possibly through the formation of an azalactone. To investigate this possibility and quantify the amount of epimerized product, a tetrapeptide FmocVal-Ala-L-Ala-Gln-NH2 and its “fully epimerized” analog, FmocVal-Ala-D-Ala-Gln-NH2, were prepared. These products would result from ligation of FmocVal-Ala-L-COSe-ortho-PhCHO with NH2-Ala-Gln-CONH2. An HPLC assay that allows detection of as low as 2% epimerization was developed. Careful analysis of HPLC traces show that detectable levels of epimerization do not occur, under the reported reaction conditions (
Condensation of FmocVal-Ala selenobenzaldehyde with NH2-Ala-Gln-CONH2 is a relatively fast reaction. To rule out the possibility that the racemization is occurring but at a slower rate, condensation of FmocAla-Val-L-COSe-o-PhCHO with NH2-Ala-Gln-CONH2was tested under the same reaction conditions. Amide bond formation involving valine selenobenzaldehyde esters is relatively slow (see Table 4 below, entries 8 and 9). Roughly 2% epimerization was observed for this slower ACL coupling (
The utility and attractiveness of native chemical ligation results from the fact that it affords chemoselective ligation of peptides, and other biomolecules, without need of protecting groups on reactive side chain functionality. The tolerance of ACL was explored for unprotected side chain groups for a variety of amino acid partners, including Arg, Asp, Cys, Glu, His, Leu, Phe, Ser, Trp, Tyr, and Val (see Table 4), was explored. These amino acid residues underwent the desired ligation, suggesting that ACL is a chemoselective reaction. Reactions of selenobenzaldehyde esters with N-terminal serine and cysteine residues may provide the amide products through the formation of respective pseudoproline intermediates, as reported for oxobenzaldehyde esters (Liu, J. Am. Chem. Soc. 116:4149-4153 (1994); Liu et al., Proc. Natl. Acad. Sci. USA 91:6584-6588 (1994); Li et al., Org. Lett. 12:1724-1727 (2010); Zhang et al., Proc. Nat'l Acad. Sci. USA 110:6657-6662 (2013), each of which is hereby incorporated by reference in its entirety), rather than through an ACL mechanism. This possibility was investigated, but the formation of the pseudoproline derivatives with seleno-benzaldehyde esters was not observed in DMF or pyridine/acetic acid mixtures (
A potential limitation of ACL is that the aldehyde group on selenobenzaldehyde esters may react with any amine, i.e., ε-amino of lysine would undergo an ACL as well as N-terminal amines (Table 4, entry 6). Possible solutions to control reactions of lysine side chain amines would include standard protecting group strategies or reactions with peptides and proteins under pH-controlled conditions. The N-terminal amino group of proteins has a significantly lower pKa value than the ε-amine of lysines (Grimsley et al., Protein Sci. 18:247-51 (2009), which is hereby incorporated by reference in its entirety). Prior studies have shown that this pKa difference can be exploited to selectively modify the N-terminal amine in the presence of lysine residues (Chan et al., J. Am. Chem. Soc. 134:2589-98 (2012), which is hereby incorporated by reference in its entirety). The possibility of selectively modifying N-terminal amines of proteins using ACL has been explored (vide infra).
Also investigated was the possibility of side chain alcohol groups of serine or tyrosine residues becoming acylated, especially with an excess of selenobenzaldehyde esters. Tripeptide NH2-ASY-CONH2 was treated with 2 or 0.5 eq of Fmoc-alanine selenobenzaldehyde ester (Table 4, entries 12-13). Acylation of the side chain alcohol group was not observed even after prolonged reaction periods. C-terminal aspartic and glutamic acid thioesters can undergo reactions with side chain carboxylates (Villain et al., Eur. J. Org. Chem. 2003:3267-72 (2003), which is hereby incorporated by reference in its entirety). This side reaction will likely also preclude placement of Asp and Glu residues, along with other reactive side chains such as cysteine, at the C-terminus in peptido selenobenzaldehydes, as in NCL.
The efficiency of peptide fragment couplings by native chemical ligation, and by other coupling agents, is significantly reduced for bulky amino acid substrates (Hackeng et al., Proc. Natl. Acad. Sci. USA 96:10068-10073 (1999), which is hereby incorporated by reference in its entirety). A range of selenobenzaldehyde esters featuring bulky C-terminal residues including valine and aminoisobutyric acid (Aib) were examined to evaluate the potential of ACL for these challenging couplings. The sterically demanding Val and Aib selenobenzaldehyde esters require longer ligation times (2-4 hours) for >95% conversion (Table 4, entries 8-10) as compared to 2-5 minutes for Ala, Gly, and Phe analogs (Table 4, entries 1-7) (Hackeng et al., Proc. Natl. Acad. Sci. USA 96:10068-73 (1999), which is hereby incorporated by reference in its entirety). Ligation of selenobenzaldehyde esters with pentapeptide (VDAFE) underwent completion in just 5 minutes (Table 4, entries 15-17), suggesting that ACL with peptides is as efficient as with single amino acid residues).
To extend the ACL technology to large peptides, a solid phase method for the synthesis of seleno-benzaldehyde esters was explored using the approach outlined by Dawson et al. for the solid phase synthesis of thioesters (
The solid phase synthesis methodology was utilized to evaluate the applicability of ACL to the synthesis of model peptides in aqueous solution. For direct comparison with NCL, sequences that have previously been explored by Dawson et al. (Hackeng et al., Proc. Natl. Acad. Sci. USA 96:10068-10073 (1999), which is hereby incorporated by reference in its entirety) were prepared. Dawson et al. noted that NCL rates of pentapeptides AcLYRAX-SPh, where X is any residue, with CRANK-CONH2 are largely dependent on the identity of X, with bulky residues (i.e., valine) at this position leading to inefficient couplings. Analogs of these pentapeptide sequences, FmocLYRAG and FmocLYRAV selenobenzaldehyde esters, were used herein to probe the effectiveness of ACL for peptide ligations with peptide FRANG-CONH2 (Table 5). As expected, ACL with glycinyl selenobenzaldehyde proceeds much faster than the analogous valinyl selenobenzaldehyde, which requires 8 hours. Yet, the ACL reaction rate with FmocLYRAV-Se-o-PhCHO is remarkable considering native chemical ligation between AcLYRAV-SPh and CRANK-CONH2 needs more than 48 hours for 60% completion (Hackeng et al., Proc. Natl. Acad. Sci. USA 96:10068-73 (1999), which is hereby incorporated by reference in its entirety). The ACL-mediated condensation of peptides in aqueous solutions is slower than in DMF, requiring roughly three times longer for completion. The HPLC analysis of crude reaction mixture shows efficient conversion of the selenoester to the desired peptide (
Acylated proline thioesters react slowly (<15% yield in >48 h) under NCL conditions (Hackeng et al., Proc. Natl. Acad. Sci. USA 96:10068-73 (1999), which is hereby incorporated by reference in its entirety). The rates of these NCL reactions have been reported to be independent of the number of residues attached to the N-terminus of proline (Pollock et al., Chem. Commun. 47:2342-44 (2011); Townsend et al., J. Am. Chem. Soc. 134:3912-16 (2012), each of which is hereby incorporated by reference in its entirety). This observation is consistent with the postulated contribution of the n-π* interaction on proline reactivity as only the carbonyl attached directly to the proline residue perturbs the rate of the reaction (Choudhary et al., Protein Sci. 20:1077-81 (2011), which is hereby incorporated by reference in its entirety). Various acylated proline derivatives were synthesized to gauge the potential of proline ligations using the ACL approach (Table 6, entries 1 and 2), and ACL was found to excel at the difficult ligation of proline residues indiscriminant of whether it is a C-terminal or N-terminal residue. Couplings of FmocVal, FmocPro, FmocAla-Pro, and FmocVal-Pro selenobenzaldehyde esters with proline methyl ester proceed rapidly, indicating that both N-terminal and C-terminal proline residues are efficient substrates for ACL (Table 6, entries 1 and 3). Analogous reactions of FmocVal and FmocPro selenophenyl esters (without the ortho-aldehyde group) require significantly longer periods (Table 6, entries 2 and 4). These studies demonstrate that preformed phenylselenoesters, which are typically considered to be highly reactive (Durek et al., Angew. Chem. Int. Ed. 50:12042-45 (2011), which is hereby incorporated by reference in its entirety), are much less efficient than the ACL auxiliary for difficult couplings.
ACL adds to a growing list of methods that allow chemoselective formation of the amide bond in aqueous buffers. The attractiveness of ACL is that it potentially allows coupling of any set of amino acid residues, without requiring specific N-terminal amino acids such as cysteine. A potentially significant limitation is that the aldehyde may capture any amine, i.e., it may be difficult to selectively modify lysine side chains or the N-terminal amines. Two solutions may be envisioned to address this potential limitation: (a) in short peptides or other synthetic oligomers and small molecules, the appropriate amine may be protected using standard approaches; (b) the pKa difference between the N-terminal amino group and the ε-amine of lysine may be exploited to achieve selective modification of the N-terminus (Grimsley et al., Protein Sci. 18:247-51 (2009); Chan et al., J. Am. Chem. Soc. 134:2589-98 (2012); Bernal-Perez et al., Anal. Biochem. 428:13-15 (2012), each of which is hereby incorporated by reference in its entirety).
The potential of ACL to specifically acylate the N-terminus of ubiquitin was evaluated (
Chemoselective reactions for amide bond formation have transformed the ability to access synthetic proteins and other bioconjugates through ligation of fragments. In these ligations, amide bond formation is accelerated by transient enforcement of an intramolecular reaction between the carboxyl and the amine termini of two fragments. A new method for peptide ligation has been introduced herein—aldehyde capture ligation, which parlays the high chemoselective reactivity of aldehydes and amines to enforce amide bond formation between amino acid residues and peptides that are difficult to ligate by existing technologies. One key feature of aldehyde capture ligation is that it employs the rapid association between an aldehyde group and an amine to enforce an intramolecular reaction leading to the desired native amide bond formation. Because primary and secondary amines react with the selenobenzaldehyde esters readily, it was postulated that the hemiaminal II (see
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow
Claims
1. A ligation agent of Formula I: fluorescent dyes, and cytotoxic small molecule drugs; represents a point of attachment to —C(O)—Se—X—(CR42)mC(O)R3; represents a point of attachment to —(CR42)mC(O)R3; represents a point of attachment to —Se—C(O)-A; and with the proviso that m is 0-2 when A is (R1)2N(C(R2)2)n— and X is
- wherein
- A is selected from the group consisting of
- X is selected from the group consisting of
- n is 1-3;
- m is 0-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine;
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- R3 is selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when A is (R1)2N(C(R2)2)n— and X is
2. The ligation agent of claim 1, wherein X is
3. The ligation agent of claim 2, wherein the ligation agent is selected from the group consisting of: wherein R3 is an aryl or an alkyl; wherein R3 is an aryl or an alkyl.
4. The ligation agent of claim 1, wherein X is
5. The ligation agent of claim 4, wherein the ligation agent is selected from the group consisting of: wherein R3 is an aryl or an alkyl.
6-15. (canceled)
16. A compound of Formula II: represents a point of attachment to —Se—Se—X—(CR42)mC(O)R3; represents a point of attachment to —(CR42)mC(O)R3; and with the proviso that m is 0-2 when X is
- wherein
- each X is selected from the group consisting of
- each
- each
- each m is independently 0-3;
- each R3 is independently selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when X is
17. The compound of claim 16, wherein X is
18. The compound of claim 17, wherein the compound is selected from the group consisting of: wherein R3 is an aryl or an alkyl; wherein R3 is an aryl or an alkyl.
19. The compound of claim 16, wherein X is
20. The compound of claim 19, wherein the compound is selected from the group consisting of: wherein R3 is an aryl or an alkyl.
21. A method of making a ligation agent of Formula I: fluorescent dyes, and cytotoxic small molecule drugs; represents a point of attachment to —C(O)—Se—X—(CR42)mC(O)R3; represents a point of attachment to —(CR42)mC(O)R3; represents a point of attachment to —Se—C(O)-A; and with the proviso that m is 0-2 when A is (R1)2N(C(R2)2)n— and X is represents a point of attachment to —Se—Se—X—(CR42)mC(O)R3; represents a point of attachment to (CR42)mC(O)R3; and fluorescent dyes, and cytotoxic small molecule drugs; represents a point of attachment to —C(O)—OH; fluorescent dyes, and cytotoxic small molecule drugs; represents a point of attachment to is a solid support;
- wherein
- A is selected from the group consisting of
- X is selected from the group consisting of
- n is 1-3;
- m is 0-3;
- each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine;
- each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains;
- R3 is selected from the group consisting of H, C1-C6 alkyl, and aryl;
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and
- R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl;
- with the proviso that m is 0-3 when A is (R1)2N(C(R2)2)n— and X is
- said method comprising: (i) providing a compound of Formula II:
- wherein each X is selected from the group consisting of
- each
- each
- each m is independently 0-3; each R3 is independently selected from the group consisting of H, C1-C6 alkyl, and aryl; each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; with the proviso that m is 0-3 when X is
- with the proviso that m is 0-2 when X is
- (ii) (a) providing a compound of Formula III:
- wherein A is selected from the group consisting of
- n is 1-3; each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine; and each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains; each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; or (ii) (b) providing a compound of Formula III′:
- wherein A is selected from the group consisting of
- Y is selected from the group consisting of O, S, Se, a good leaving group, and Dawson's auxiliary; Y′ is optionally present and, if present, is a C1-C6 alkyl, an ester, or an amide;
- n is 1-3; each R1 is independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, amino acids, peptides, and protecting groups for the protection of an amine; and each R2 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, —C(O)NR5R6, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, C1-C6 alkoxy, and amino acid side chains; each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy; and R5, R6, R7, and R8 are each independently selected from the group consisting of H, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, and aryl; and (iii) reacting the compound of Formula II with the compound of Formula III or the compound of Formula III′ under conditions effective to make a ligation agent of Formula I.
22. The method of claim 21, wherein said providing a compound of Formula II comprises:
- providing a substituted halogenated benzene aldehyde or substituted halogenated benzene ketone; and
- reacting the substituted halogenated benzene aldehyde or substituted halogenated benzene ketone with a diselenide in the presence of a cation scavenger under conditions effective to produce a compound of Formula II.
23. The method of claim 22, wherein the substituted halogenated benzene aldehyde or substituted halogenated benzene ketone is a compound of Formula IV: represents a point of attachment to —X′; represents a point of attachment to —(CR42)mC(O)R3;
- wherein
- X is selected from the group consisting of
- X′ is a halogen;
- each m is independently 0-3;
- each R3 is independently selected from the group consisting of H, C1-C6 alkyl, and aryl; and
- each R4 is independently selected from the group consisting of H, —C(O)R7, —C(O)OR8, NO2, —NR5R6, halogen, OH, C1-C6 alkyl, aryl, and C1-C6 alkoxy.
24. The method of claim 22, wherein the diselenide is an alkaline metal diselenide.
25. The method of claim 22, wherein the cation scavenger is selected from the group consisting of HMPA, DMPU, DMI, tetraalkyl ureas, and cyclic alkyl ureas.
26. The method of claim 21, wherein a compound of Formula III is provided.
27. The method of claim 21, wherein a compound of Formula III′ is provided.
28. The method of claim 27, wherein the solid support is selected from the group consisting of a Tenta gel, Rink amide resin, Merrifield resin, trityl resin, PAM resin, and Kaiser resin.
Type: Application
Filed: Mar 13, 2017
Publication Date: Aug 31, 2017
Inventors: Paramjit S. Arora (Cold Spring Harbor, NY), Monika Raj (Jersey City, NJ), Huabin Wu (Ningde City)
Application Number: 15/457,220
