1. A composition of purified homogeneously glycosylated erythropoietin or a purified homogenously glycosylated fragment thereof.
2. The composition of claim 1, wherein the primary amino acid sequence of the erythropoietin is as follows:
(SEQ ID NO: 1)
Ala-Pro-Pro-Arg-Leu-Ile-Cys-Asp-Ser-Arg-Val-Leu-
Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-Glu-Ala-Glu-Asn-
Ile-Thr-Thr-Gly-Cys-Ala-Glu-His-Cys-Ser-Leu-Asn-
Glu-Asn-Ile-Thr-Val-Pro-Asp-Thr-Lys-Val-Asn-Phe-
Tyr-Ala-Trp-Lys-Arg-Met-Glu-Val-Gly-Gln-Gln-Ala-
Val-Glu-Val-Trp-Gln-Gly-Leu-Ala-Leu-Leu-Ser-Glu-
Ala-Val-Leu-Arg-Gly-Gln-Ala-Leu-Leu-Val-Asn-Ser-
Ser-Gln-Pro-Trp-Glu-Pro-Leu-Gln-Leu-His-Val-Asp-
Lys-Ala-Val-Ser-Gly-Leu-Arg-Ser-Leu-Thr-Thr-Leu-
Leu-Arg-Ala-Leu-Gly-Ala-Gln-Lys-Glu-Ala-Ile-Ser-
Pro-Pro-Asp-Ala-Ala-Ser-Ala-Ala-Pro-Leu-Arg-Thr-
Ile-Thr-Ala-Asp-Thr-Phe-Arg-Lys-Leu-Phe-Arg-Val-
Tyr-Ser-Asn-Phe-Leu-Arg-Gly-Lys-Leu-Lys-Leu-Tyr-
Thr-Gly-Glu-Ala-Cys-Arg-Thr-Gly-Asp-Arg.
3. The composition of claim 1, wherein the erythropoietin is glycosylated at Asn24, Asn38, Asn83, and Ser126.
4. The composition of claim 1, wherein the primary amino acid sequence of the fragment of erythropoietin is as follows:
(Amino acids 1-28; SEQ ID NO: 3)
Ala-Pro-Pro-Arg-Leu-Ile-Cys-Asp-Ser-Arg-Val-Leu-
Glu-Arg-Tyr-Leu-Leu-Glu-Ala-Lys-Glu-Ala-Glu-Asn-
Ile-Thr-Thr-Gly;
(Amino acids 29-77; SEQ ID NO: 4)
Cys-Ala-Glu-His-Cys-Ser-Leu-Asn-Glu-Asn-Ile-Thr-
Val-Pro-Asp-Thr-Lys-Val-Asn-Phe-Tyr-Ala-Trp-Lys-
Arg-Met-Glu-Val-Gly-Gln-Gln-Ala-Val-Glu-Val-Trp-
Gln-Gly-Leu-Ala-Leu-Leu-Ser-Glu-Ala-Val-Leu-Arg-
Gly;
(Amino acids 78-113; SEQ ID NO: 5)
Gln-Ala-Leu-Leu-Val-Asn-Ser-Ser-Gln-Pro-Trp-Glu-
Pro-Leu-Gln-Leu-His-Val-Asp-Lys-Ala-Val-Ser-Gly-
Leu-Arg-Ser-Leu-Thr-Thr-Leu-Leu-Arg-Ala-Leu-Gly;
or
(Amino acids 114-166; SEQ ID NO: 6)
Ala-Gln-Lys-Glu-Ala-Ile-Ser-Pro-Pro-Asp-Ala-Ala-
Ser-Ala-Ala-Pro-Leu-Arg-Thr-Ile-Thr-Ala-Asp-Thr-
Phe-Arg-Lys-Leu-Phe-Arg-Val-Tyr-Ser-Asn-Phe-Leu-
Arg-Gly-Lys-Leu-Lys-Leu-Tyr-Thr-Gly-Glu-Ala-Cys-
Arg-Thr-Gly-Asp-Arg.
5. The composition of claim 1, wherein the structure of the erythropoietin is of formula:
6. The composition of claim 1 wherein the structure of the fragment of erythropoietin is as follows:
7. The composition of claim 1 wherein the glycosylation pattern is identical on all erythropoietin molecules or fragments thereof of the composition.
8. A method of preparing homogeneously glycosylated erythropoietin, the method comprising steps of:
ligating the glycosylated fragments EPO (1-28); EPO (29-77); EPO (78-113); and EPO (114-166) together to form a homogeneously glycosylated erythropoietin.
9-34. (canceled)
35. An isolated glycopeptide having formula:
wherein the peptide has an amino acid sequence that is either identical to or closely related to that of a naturally occurring glycoprotein near a glycosylation site, or a truncated, elongated or derivatized version thereof; wherein any one or more of the amino acid residues may bear one or more protecting groups;
each occurrence of L1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety; or a natural or non-natural amino acid side chain; and
RX2 is ORX0, ORX2a, \u2014SRX2a, or \u2014NRX2bRX2c, wherein RX2a is hydrogen, alkyl, aromatic, heteroaromatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a carboxylic acid protecting group, an amino acid, or a protected amino acid; and RX2b and RX2c are independently hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a nitrogen protecting group, an amino acid or a protected amino acid;
RX0 is a group such that the moiety \u2014C(\u2550O)ORX0 can be made to undergo ligation with a peptide acyl acceptor;
P1 is hydrogen, a nitrogen protecting group, or a moiety having the structure:
wherein n is 1, 2, 3, or 4; RS1 is hydrogen; substituted or unsubstituted aryl;
substituted or unsubstituted heteroaryl; or a sulfide protecting group; each occurrence of RB is independently alkoxy, hydroxy, or silyloxy; and
A is a carbohydrate;
with the proviso that the glycopeptide is not a naturally occurring glycopeptide.
36. The glycopeptide of claim 35, wherein P1 is a moiety having the structure:
and n is 2.
37. The glycopeptide of claim 35, wherein P1 is a moiety having the structure:
and n is 3.
38. The glycopeptide of claim 35, wherein P1 is an Fmoc nitrogen protecting group.
39. The glycopeptide of claim 35, wherein P1 is hydrogen.
40. The glycopeptide of claim 35, wherein P1 is
41. The glycopeptide of claim 35, wherein P1 is
42. The glycopeptide of claim 35, wherein RX2 is \u2014ORX2a, wherein RX2a is
43. The glycopeptide of claim 35, wherein RX2 is \u2014ORX2a, wherein RX2a is C1-C6 alkyl.
44. The glycopeptide of claim 35, wherein RX2 is \u2014ORX2a, wherein RX2a is a carboxylic acid protecting group.
45. The glycopeptide of claim 35, wherein RX2 is \u2014OH.
46. The glycopeptide of claim 35, wherein RX2 is \u2014SRX2a.
47. The glycopeptide of claim 35 having the structure:
48. The glycopeptide of claim 35 having the structure:
49. The glycopeptide of claim 35 having the structure:
50. The glycopeptide of claim 35 having the structure:
51. An isolated intermediate having the structure:
wherein
the peptide backbone comprises two or more amino acids;
X is N or CH;
n is 2 or 3;
RA1 and RA2 are independently natural or non-natural amino acid side chains;
each occurrence of RB is independently alkoxy, hydroxy or silyloxy;
k1 and k2 are independently integers between 1 and about 20;
each occurrence of A1 and A2 is independently an aliphatic, heteroaliphatic, aromatic, heteroaromatic, aryl, heteroaryl, carbohydrate, or a pharmaceutically useful group or entity;
RX0 is a group such that the moiety \u2014C(\u2550O)ORX0 can be made to undergo ligation with a peptide acyl acceptor;
each occurrence of L1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety or a natural or non-natural amino acid side chain; and
RX2 is \u2014ORX2a, \u2014SRX2a, or \u2014NRX2bRX2c, wherein RX2a is hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a carboxylic acid protecting group, an amino acid or a protected amino acid; and RX2b and RX2c are independently hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a nitrogen protecting group, an amino acid or a protected amino acid.
52. The intermediate of claim 51, wherein RX2 is \u2014ORX2a, wherein RX2a is
53. The intermediate of claim 51, wherein RX2 is \u2014ORX2a, wherein RX2a is C1-C6 alkyl.
54. The intermediate of claim 51, wherein RX2 is \u2014ORX2a wherein RX2a is a carboxylic acid protecting group.
55. The intermediate of claim 51, wherein RX2 is \u2014OH.
56. The intermediate of claim 51, wherein RX2 is \u2014SRX2a.
57. The intermediate of claim 51 which is prepared by reacting:
under suitable conditions.
58. The intermediate of claim 51 having the structure:
59. The intermediate of claim 51 having the structure:
60. The intermediate of claim 59 having the structure:
61-62. (canceled)
63. A peptide of formula:
wherein
Peptide1 is a peptide comprising two or more natural or unnatural amino acids, wherein the peptide is protected, partially protected, or unprotected, and the peptide is optionally glycosylated;
n is 0, 1, 2, 3, or 4;
RA is hydrogen; a substituted or unsubstituted, linear or branched, cyclic or acyclic saturated or unsaturated aliphatic; a substituted or unsubstituted, linear or branched, cyclic or acyclic saturated or unsaturated heteroaliphatic; substituted or unsubstituted aryl; or substituted or unsubstituted heteroaryl;
each occurrence of RB is independently hydrogen; halogen; alkoxy; \u2014CN; \u2014NO2;
substituted or unsubstituted acyl; a substituted or unsubstituted, linear or branched, cyclic or acyclic saturated or unsaturated aliphatic, or a substituted or unsubstituted, linear or branched, cyclic or acyclic saturated or unsaturated heteroaliphatic; and
RC1 is a side chain of a natural or unnatural amino acid.
64. The peptide of claim 63, wherein the peptide has at least one post-translational modification.
65. The peptide of claim 63, wherein the peptide is glycosylated.
66. The peptide of claim 63, wherein RC1 is a side chain of a natural amino acid.
67. The peptide of claim 63, wherein the peptide has an unprotected N-terminus.
68. The peptide of claim 63, wherein the peptide has a protected N-terminus.
69. The peptide of claim 68, wherein the peptide has an Fmoc-protected N-terminus.
70. A method for ligating two peptides, wherein each peptide comprises a peptidic backbone made up of two or more amino acids wherein one or more amino acids are independently substituted with a moiety having the structure,
the method comprising steps of:
coupling a peptide acyl donor comprising a peptidic backbone made up of two or more amino acids wherein said peptide acyl donor has the structure:
with a peptide acyl acceptor comprising a peptidic backbone made up of two or more amino acids wherein said peptide acyl acceptor has the structure:
under suitable conditions to form the adduct of formula:
wherein
n is 2 or 3;
RA1 and RA2 are independently natural or non-natural amino acid side chains;
each occurrence of RB is independently alkoxy, hydroxy, or silyloxy;
k1 and k2 are independently integers between 1 and 20, inclusive;
each occurrence of A1 and A2 is independently an aliphatic, heteroaliphatic, aromatic, heteroaromatic, aryl, heteroaryl, carbohydrate, or a pharmaceutically useful group or entity;
RS2 is hydrogen;
RX0 is a group such that the moiety \u2014C(\u2550O)ORX0 can be made to undergo ligation with the peptide acyl acceptor;
each occurrence of L1 and L2 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety or a natural or non-natural amino acid side chain; and
RX2 is \u2014ORX2a or \u2014NRX2bRX2c, wherein RX2a is hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a carboxylic acid protecting group, an amino acid or a protected amino acid; and RX2b and RX2c are independently hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a nitrogen protecting group, an amino acid or a protected amino acid.
71-73. (canceled)
74. A method for preparing a polyfunctionalized peptideprotein comprising a peptidic backbone made up of four or more amino acids wherein two or more non-adjacent amino acids are independently substituted with a moiety having the structure:
wherein the method comprises steps of:
(a) coupling a peptide acyl donor comprising a peptidic backbone made up of two or more amino acids wherein said peptide acyl donor has the structure:
with a starting peptide acyl acceptor comprising a peptidic backbone made up of two or more amino acids wherein said peptide acyl acceptor has the structure:
under suitable conditions to form a resulting peptide acyl acceptor having the structure:
(b) repeating step (a) using the resulting peptide acyl acceptor of step (a) as starting peptide acyl acceptor to give a peptideprotein having the structure:
(c) deprotecting the polyfunctionalized peptideprotein of step (b) to give a peptideprotein having the structure:
or a salt form thereof,
wherein a is an integer between 1 and 20, inclusive;
each occurrence of n is independently 2 or 3;
each occurrence of RA1 and RA2 is independently a natural or non-natural amino acid side chain;
each occurrence of RB is independently alkoxy, hydroxy, or silyloxy;
each occurrence of k1 and k2 is independently an integer between 1 and about 20;
each occurrence of A, A1 and A2 is independently an aliphatic, heteroaliphatic, aromatic, heteroaromatic, aryl, heteroaryl or a pharmaceutically useful group or entity;
RS1 is hydrogen or a sulfide protecting group;
RX0 is a group such that the moiety \u2014C(\u2550O)ORX0 can be made to undergo ligation with the peptide acyl acceptor;
each occurrence of L1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety or a natural or non-natural amino acid side chain; and
RX2 is \u2014ORX2a or \u2014NRX2bRX2c, wherein RX2a is hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a carboxylic acid protecting group, an amino acid or a protected amino acid; and RX2b and RX2c are independently hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a nitrogen protecting group, an amino acid or a protected amino acid.
75-96. (canceled)
97. A method for preparing a cyclic peptide having the structure:
said method comprising a step of subjecting a peptide having the structure:
to suitable conditions to effect ligation;
wherein the peptide comprises at least four amino acid residues;
n is 2 or 3;
each occurrence of RB is independently alkoxy, hydroxy or silyloxy;
k1 is an integer between 0 and about 20;
each occurrence of A is independently an aliphatic, heteroaliphatic, aromatic, heteroaromatic, aryl, heteroaryl or a pharmaceutically useful group or entity;
RS1 is hydrogen or a sulfide protecting group;
RX0 is a group such that the moiety \u2014C(\u2550O)ORX0 can be made to undergo ligation with the N-terminal peptide acyl acceptor; and
each occurrence of L1 is independently a substituted or unsubstituted, linear or branched, cyclic or acyclic, saturated or unsaturated aliphatic or heteroaliphatic moiety or a natural or non-natural amino acid side chain.
98-121. (canceled)
122. A method of desulfurizing or deselenizing a peptide, the method comprising steps of:
desulfurzing or deselenizing a peptide of formula:
wherein
Peptide1 is a peptide comprising two or more natural or unnatural amino acids, wherein the peptide is protected, partially protected, or unprotected, and the peptide is optionally glycosylated;
Peptide2 is a peptide comprising two or more natural or unnatural amino acids, wherein the peptide is protected, partially protected, or unprotected, and the peptide is optionally glycosylated;
X is Se or S;
RC1 is a side chain of a natural or unnatural amino acid, wherein the side chain is protected or unprotected, and the side chain is optionally glycosylated;
under suitable conditions to form a peptide of formula:
123-135. (canceled)
136. A peptide of formula:
wherein
each occurrence of RN, RC, and R is independently the side chain of a natural or unnatural amino acid, optionally glycosylated;
PC is \u2014ORX2a, \u2014SRX2a, or \u2014NRX2bRX2c, wherein RX2a is hydrogen, alkyl, aromatic, heteroaromatic, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -alkyl(aryl), -alkyl (heteroaryl), a carboxylic acid protecting group, an amino acid, or a protected amino acid; and RX2b and RX2c are independently hydrogen, alkyl, aromatic, heteroaromatic, aryl, heteroaryl, -alkyl(aryl), -alkyl(heteroaryl), a nitrogen protecting group, an amino acid or a protected amino acid;
PN is hydrogen, a nitrogen protecting group, or a moiety having the structure:
wherein n is 1, 2, 3, or 4; RS1 is hydrogen; substituted or unsubstituted aryl;
substituted or unsubstituted heteroaryl; or a sulfide protecting group; each occurrence of RB is independently alkoxy, hydroxy, or silyloxy.
137-150. (canceled)
151. The glycopeptide of claim 35, wherein A is O-linked or N-linked, and A is selected from the group consisting of:
when A is O-linked; and
when A is N-linked.
152. The intermediate of claim 51, wherein A1 and A2 are independently O-linked or N-linked, and A1 and A2 are independently selected from the group consisting of:
when A1 or A2 is O-linked; and
when A1 or A2 is N-linked.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A method for mapping a Universal Fleet Member Identifier (UFMI) to a previously assigned UFMI, comprising the steps of:
receiving an Integrated Digital Enhanced Network (iDEN) routing request message from a dispatch application processor (DAP) associated with a calling party at a serving home location register (HLR) of a called party, wherein the routing request message includes a first UFMI associated with the called party;
obtaining a second UFMI of the called party by the HLR by consulting a subscriber table stored in the HLR;
transmitting an iDEN routing response message to the DAP by the HLR, wherein the routing response message includes the second UFMI associated with the called party;
determining a second HLR associated with the called party by the DAP by using the second UFMI, wherein the second HLR is associated with a gateway from an iDEN network;
transmitting a second iDEN routing request message from the DAP to the gateway, wherein the second routing request message includes the second UFMI; and
transmitting a second iDEN routing response message to the DAP by the gateway.
2. The method of claim 1 wherein the newly assigned UFMI is a same number as an interconnect phone number associated with the called party.
3. The method of claim 1 further comprising the step of provisioning the called party as an external subscriber in the HLR.
4. The method of claim 1 wherein the routing response message also includes a parameter identifying a current visited location register (VLR) associated with the called party.
5. The method of claim 4 wherein the current visited location register is associated with a gateway from an iDEN network.
6. The method of claim 1 wherein the subscriber table includes the previously assigned UFMI, the newly assigned UFMI, and a parameter identifying a current visited location register (VLR) associated with the called party, and wherein the table associates the previously assigned UFMI to the newly assigned UFMI and to the parameter identifying the current VLR.
7. The method of claim 1 wherein the previously assigned UFMI is associated with an iDEN network and the newly assigned UFMI is associated with a next generation network.
8. The method of claim 7 wherein the next generation network is a Code Division Multiple Access (CDMA) network.
9. The method of claim 7 wherein the next generation network uses Push-to-Talk over Cellular (PoC).
10. The method of claim 5 further comprising the step of receiving a private call request forward message at the gateway from the DAP, wherein the private call request forward message includes the newly assigned UFMI.
11. A system for mapping a Universal Fleet Member Identifier (UFMI) to a previously assigned UFMI, comprising:
an Integrated Digital Enhanced Network (iDEN) including:
a dispatch application processor (DAP) associated with a calling party; and
a home location register (HLR) associated with a called party and including a subscriber table;
wherein an iDEN routing request message that includes a first UFMI associated with the called party is received from the DAP at the HLR, wherein the HLR obtains a second UFMI associated with the called party by consulting the subscriber table, and wherein the HLR transmits an iDEN routing response message that includes the second UFMI to the DAP:
and further wherein the DAP determines a second HLR associated with the called party by using the second UFMI and wherein the second HLR is associated with a gateway from the iDEN network.
12. The system of claim 11 wherein the routing response message also includes a parameter identifying a current visited location register (VLR) associated with the called party.
13. The system of claim 12 wherein the current visited location register is associated with a gateway from the iDEN network.
14. The system of claim 11 wherein the subscriber table includes the first UFMI, the second UFMI, and a parameter identifying a current visited location register (VLR) associated with the called party, and wherein the table associates the first UFMI to the second UFMI and to the parameter identifying the current VLR.
15. The system of claim 11 wherein the first UFMI is associated with the iDEN network and second UFMI is associated with a next generation network.
16. The system of claim 15 wherein the next generation network is a Code Division Multiple Access (CDMA) network.
17. The system of claim 15 wherein the next generation network uses Push-to-Talk over Cellular (PoC).
18. The system of claim 11 wherein the DAP transmits a second iDEN routing request message to the gateway and wherein the second routing request message includes the second UFMI.
19. The system of claim 18 wherein the gateway transmits a second iDEN routing response message to the DAP.