U.S. patent application number 11/340730 was filed with the patent office on 2006-07-06 for pseudo-native chemical ligation.
This patent application is currently assigned to Amylin Pharmaceuticals, Inc.. Invention is credited to Paolo Botti, James A. Bradburne, Shiah-yun Chen, Sonya Cressman, Christie L. Hunter, Stephen B.H. Kent, Gerd G. Kochendoerfer, Donald W. Low.
Application Number | 20060149039 11/340730 |
Document ID | / |
Family ID | 26925033 |
Filed Date | 2006-07-06 |
United States Patent
Application |
20060149039 |
Kind Code |
A1 |
Hunter; Christie L. ; et
al. |
July 6, 2006 |
Pseudo-native chemical ligation
Abstract
The present invention concerns methods and compositions for
extending the technique of native chemical ligation to permit the
ligation of a wider range of peptides, polypeptides, other polymers
and other molecules via an amide bond. The invention further
provides methods and uses for such proteins and derivatized
proteins. The invention is particularly suitable for use in the
synthesis of optionally polymer-modified, synthetic bioactive
proteins, and of pharmaceutical compositions that contain such
proteins.
Inventors: |
Hunter; Christie L.; (San
Mateo, CA) ; Botti; Paolo; (Placenza, IT) ;
Bradburne; James A.; (Redwood City, CA) ; Chen;
Shiah-yun; (Mountain View, CA) ; Cressman; Sonya;
(Ladysmith, CA) ; Kent; Stephen B.H.; (San
Francisco, CA) ; Kochendoerfer; Gerd G.; (Oakland,
CA) ; Low; Donald W.; (Burlingame, CA) |
Correspondence
Address: |
The Chandler Law Firm
10621 River Road
Potomac
MD
20854
US
|
Assignee: |
Amylin Pharmaceuticals,
Inc.
San Diego
CA
|
Family ID: |
26925033 |
Appl. No.: |
11/340730 |
Filed: |
January 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10332386 |
Jan 8, 2003 |
7030218 |
|
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PCT/US01/21935 |
Jul 12, 2001 |
|
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11340730 |
Jan 27, 2006 |
|
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60231339 |
Sep 8, 2000 |
|
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60236377 |
Sep 29, 2000 |
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Current U.S.
Class: |
530/351 ;
530/350; 530/395 |
Current CPC
Class: |
A61P 37/06 20180101;
A61P 9/10 20180101; A61P 19/02 20180101; A61P 29/00 20180101; A61P
17/00 20180101; A61P 17/02 20180101; A61P 43/00 20180101; A61P
11/06 20180101; A61P 37/08 20180101; A61P 31/12 20180101; A61P
11/02 20180101; C07K 14/505 20130101; A61P 37/00 20180101; A61K
38/00 20130101; A61P 7/00 20180101; A61P 7/06 20180101 |
Class at
Publication: |
530/351 ;
530/350; 530/395 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61K 38/18 20060101 A61K038/18; C07K 14/53 20060101
C07K014/53; C07K 14/51 20060101 C07K014/51 |
Claims
1. A synthetic protein containing a pseudo-amino acid residue whose
side chain has the formula: --S--R.sub.aa, where R.sub.aa is an
optionally substituted terminal portion of a ribosomally-specified
amino acid side chain, or an analog of said terminal portion of a
ribosomally-specified amino acid side chain.
2. The synthetic protein of claim l, wherein said side chain
--S--R.sub.aa has the same chain length as the side chain of said
ribosomally-specified amino acid.
3. The synthetic protein of claim 1, wherein said side chain
--S--R.sub.aa has a greater chain length than the side chain of
said ribosomally-specified amino acid.
4. The synthetic protein of claim 1, wherein said protein has a
biological activity possessed by a ribosomally-specified bioactive
protein.
5. The synthetic protein of claim 4, wherein said side chain
--S--R.sub.aa of said pseudo amino acid residue mimics the
structure or function of a ribosomally-specified amino acid at a
corresponding position in said bioactive protein.
6. The synthetic protein of claim 4, wherein said side chain
--S--R.sub.aa of said pseudo amino acid residue alters the
structure or function of a ribosomally-specified amino acid at a
corresponding position in said bioactive protein.
7. The synthetic protein of claim 4, wherein said synthetic protein
has a monomer molecular weight greater than about 25 kDa.
8. The synthetic protein of claim 4, wherein said ribosomally
produced bioactive protein contains a cysteine residue.
9. The synthetic protein of claim 8, wherein said pseudo amino acid
residue is at a position other than one corresponding to the
position of said cysteine residue in said ribosomally produced
bioactive protein.
10. The synthetic protein of claim 1, wherein a plurality of amino
acid residues of said protein are bound to adjacent amino acid
residues by an amide bond.
11. The synthetic protein of claim 10, wherein each amino acid
residue is bonded to its adjacent amino acid residues by amide
bonds.
12. The synthetic protein of claim 1, wherein said protein is
composed of a plurality of amino acid residues wherein at least one
of such amino acid residues is bonded to an adjacent amino acid
residue by a non-amide bond.
13. The synthetic protein of claim 12, wherein said non-amide bond
is selected from the group consisting of a thioester bond, a
thioether bond, and an oxime bond.
14. The synthetic protein of claim 1, wherein said pseudo-amino
acid residue is a D-configuration amino acid residue.
15. The synthetic protein of claim 1, wherein said pseudo-amino
acid residue is an L-configuration amino acid residue.
16. The synthetic protein of claim 1, wherein said pseudo-amino
acid residue is selected from the group consisting of a
pseudo-arginine; a pseudo-asparagine; a pseudo-aspartate; a
pseudo-dopamine; a pseudo-glutamate; a pseudo-glutamine; a
pseudo-histidine; a pseudo-isoleucine; a pseudo-leucine; a
pseudo-lysine; a pseudo-methionine; a pseudo-phenyalanine; a
pseudo-serine; a pseudo-threonine; a pseudo-tryptophan; a
pseudo-tyrosine; and a pseudo-valine.
17. The synthetic protein of claim 16 wherein said pseudo-amino
acid residue is a pseudo-glutamate.
18. The synthetic protein of claim 4, wherein said ribosomally
produced bioactive protein is a mammalian protein.
19. The synthetic protein of claim 18, wherein said mammalian
protein is selected from the group consisting of a human, simian,
bovine, murine, porcine, ovine, and equine protein.
20. The synthetic protein of claim 18, wherein said mammalian
protein is a human protein.
21. The synthetic protein of any of claims 19-20, wherein said
protein has a bioactivity selected of a protein receptor or
fragment thereof, of a protein receptor ligand or fragment thereof,
or of a cytokine.
22. The synthetic protein of claim 21, wherein said synthetic
bioactive protein has a bioactivity of a cytokine.
23. The synthetic protein of claim 22, wherein said cytokine is
selected from the group consisting of an interleukin, a lymphokine,
a RANTES protein, an erythropoiesis stimulating protein, tumor
necrosis factor (TNF), an interferon, a growth factors and a single
peptide hormone.
24. The synthetic protein of claim 23, wherein said cytokine is an
erythropoiesis stimulating protein.
25. The synthetic protein of claim 24, wherein said erythropoiesis
stimulating protein is erythropoietin.
26. The synthetic protein of claim 25, wherein said synthetic
bioactive protein is selected from the group consisting of SEP-0,
SEP-1, and SEP-3.
27. The synthetic protein of claim 22, wherein said cytokine is a
RANTES protein.
28. The synthetic protein of claim 22, wherein said cytokine is a
growth factor.
29. The synthetic protein of claim 28, wherein said growth factor
is G-CSF.
30. The synthetic protein of claim 1, wherein said synthetic
protein comprises one or more amino acid residues that are modified
by one or more polymer adducts.
31. The synthetic protein of claim 4, wherein said ribosomally
produced bioactive protein is glycosylated at one or more
glycosylation sites.
32. The synthetic protein of claim 31, wherein said synthetic
protein comprises one or more amino acid residues that are modified
by one or more polymer adducts at amino acid residue(s) that
correspond to at least one of said glycosylation sites of said
ribosomally produced bioactive protein.
33. A molecularly homogeneous pharmaceutical composition comprising
a synthetic protein, wherein said protein possesses a biological
activity that mimics a biological activity associated with a
ribosomally-specified bioactive mammalian protein, and has a
monomer molecular weight greater than about 25 kDa, and contains a
pseudo-amino acid residue whose side chain has the formula:
--S--R.sub.aa, where R.sub.aa is selected from the group consisting
of an optionally substituted terminal portion of a
ribosomally-specified amino acid side chain, or an analog
thereof.
34. The pharmaceutical composition of claim 33, wherein said
composition comprises a mixture of at least two of said molecularly
homogeneous pharmaceutical compositions.
35. The pharmaceutical composition of claim 33, wherein said
ribosomally-specified bioactive mammalian protein is selected from
the group consisting of a human, simian, bovine, murine, porcine,
ovine, and equine protein.
36. The pharmaceutical composition of claim 33, wherein said
ribosomally-specified bioactive mammalian protein is a human
protein.
37. The pharmaceutical composition of claim 36, wherein said
synthetic protein has a biological activity of a protein receptor
or fragment thereof, of a protein receptor ligand or fragment
thereof, or of a cytokine.
38. The pharmaceutical composition of claim 37, wherein said
synthetic protein has a biological activity of a cytokine.
39. The pharmaceutical composition of claim 38, wherein said
cytokine is selected from the group consisting of an interleukin, a
RANTES protein, a lymphokine, an erythropoiesis stimulating
protein, tumor necrosis factor (TNF), an interferon, a growth
factor and a single peptide hormone.
40. The pharmaceutical composition of claim 39, wherein said
cytokine is an erythropoiesis stimulating protein.
41. The pharmaceutical composition of claim 40, wherein said
erythropoiesis stimulating protein is erythropoietin.
42. The pharmaceutical composition of claim 41, wherein said
synthetic protein is selected from the group consisting of SEP-0,
SEP-1, and SEP-3.
43. The pharmaceutical composition of claim 38, wherein said
cytokine is a RANTES protein.
44. The pharmaceutical composition of claim 39, wherein said
cytokine is a growth factor.
45. The pharmaceutical composition of claim 44, wherein said growth
factor is G-CSF.
46. A method of treating a human disease or condition that
comprises administering to an individual in need of such treatment
an effective amount of a pharmaceutical composition comprising one
or more molecularly homogeneous pharmaceutical compositions each
comprising a synthetic protein, wherein said synthetic protein has
a monomer molecular weight greater than about 25 kDa, and contains
a pseudo-amino acid residue whose side chain has the formula:
--S--R.sub.aa where R.sub.aa is selected from the group consisting
of an optionally substituted terminal portion of a
ribosomally-specified amino acid side chain, or an analog thereof;
said synthetic protein possessing a biological activity that mimics
a biological activity of a ribosomally-specified bioactive human
protein receptor or fragment thereof, protein receptor ligand or
fragment thereof, or a cytokine.
47. The method of claim 46, wherein said synthetic protein has a
biological activity of a cytokine.
48. The method of claim 47, wherein said cytokine is selected from
the group consisting of an interleukin, a lymphokine, a RANTES
protein, an erythropoiesis stimulating protein, tumor necrosis
factor (TNF), an interferon, a growth factor and a single peptide
hormone.
49. The method of claim 48, wherein said cytokine is an
erythropoiesis stimulating protein.
50. The method of claim 49, wherein said erythropoiesis stimulating
protein is erythropoietin.
51. The method of claim 50, wherein said synthetic bioactive
protein is selected from the group consisting of SEP-0, SEP-1, and
SEP-3.
52. The method of claim 48, wherein said cytokine is a RANTES
protein.
53. The method of claim 48, wherein said cytokine is a growth
factor.
54. The method of claim 53, wherein said growth factor is
G-CSF.
55. A method for synthesizing a desired polypeptide of formula:
aa.sub.NH.sub.2-Q-aa.sub.x-aa.sub.y-W-aa.sub.COOH where Q and W
each denote the optional presence of one or more additional amino
acid residues, aa.sub.NH.sub.2 denotes the N-terminal amino acid
residue of the polypeptide; aa.sub.x and aa.sub.y denote internal
adjacent amino acid residues, having side chains x and y,
respectively, and aa.sub.COOH denotes the C-terminal amino acid
residue of the polypeptide; said method comprising: (A) ligating a
first peptide having the formula: aa.sub.NH.sub.2-Q-aa.sub.x-COSR,
wherein R is any group compatible with the thioester group,
including, but not limited to, aryl, benzyl, and alkyl groups, to a
second peptide having the formula: Cys-W-aa.sub.COOH to thereby
form the polypeptide: aa.sub.NH.sub.2-Q-aa.sub.x-Cys-W-aa.sub.COOH;
and (B) incubating said polypeptide in the presence of a reagent
R.sub.aa--X, where R.sub.aa is a group whose structure mimics the
terminal portion of the side chain of amino acid residue aa.sub.y;
and X is a good leaving group; said incubation being under
conditions sufficient to form said desired polypeptide.
56. The method of claim 55, wherein X is a halogen.
57. The method of claim 56, wherein said halogen is F, I or Br.
58. The method of claim 55, wherein said amino acid residue
aa.sub.y is selected from the group consisting of a
pseudo-arginine; a pseudo-asparagine; a pseudo-aspartate; a
pseudo-dopamine; a pseudo-glutamate; a pseudo-glutamine; a
pseudo-histidine; a pseudo-isoleucine; a pseudo-leucine; a
pseudo-lysine; a pseudo-methionine; a pseudo-phenyalanine; a
pseudo-serine; a pseudo-threonine; a pseudo-tryptophan; a
pseudo-tyrosine; and a pseudo-valine.
59. A polypeptide of formula:
aa.sub.NH.sub.2-Q-aa.sub.x-aa.sub.y-W-aa.sub.COOH where Q and W
each denote the optional presence of one or more additional amino
acid residues, aa.sub.NH.sub.2 denotes the N-terminal amino acid
residue of the polypeptide; aa.sub.x and aa.sub.y denote internal
adjacent amino acid residues, having side chains x and y,
respectively, and aa.sub.COOH denotes the C-terminal amino acid
residue of the polypeptide; wherein said polypeptide is produced by
a method comprising: (A) ligating a first peptide having the
formula: aa.sub.NH.sub.2-Q-aa.sub.x-COSR, wherein R is any group
compatible with the thioester group, including, but not limited to,
aryl, benzyl, and alkyl groups, to a second peptide having the
formula: Cys-W-aa.sub.COOH to thereby form the polypeptide:
aa.sub.NH.sub.2-Q-aa.sub.x-Cys-W-aa.sub.COOH; and (B) incubating
said polypeptide in the presence of a reagent R.sub.aa--X, where
R.sub.aa is a group whose structure mimics the terminal portion of
the side chain of amino acid residue aa.sub.y; and X is a good
leaving group; said incubation being under conditions sufficient to
form said desired polypeptide.
60. The polypeptide of claim 59, wherein X is a halogen.
61. The polypeptide of claim 60, wherein said halogen is F, I or
Br.
62. The polypeptide of claim 59, wherein said amino acid residue
aa.sub.y is selected from the group consisting of a
pseudo-arginine; a pseudo-asparagine; a pseudo-aspartate; a
pseudo-dopamine; a pseudo-glutamate; a pseudo-glutamine; a
pseudo-histidine; a pseudo-isoleucine; a pseudo-leucine; a
pseudo-lysine; a pseudo-methionine; a pseudo-phenyalanine; a
pseudo-serine; a pseudo-threonine; a pseudo-tryptophan; a
pseudo-tyrosine; and a pseudo-valine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/332,386 (filed Jan. 8 2003) and
PCT/US01/21935, and claims priority to U.S. Patent Applications
Ser. Nos. 60/231,339 (filed Sep. 8, 2000) and 60/236,377 (filed
Sep. 29, 2000), all of which applications are herein incorporated
by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions
for extending the technique of native chemical ligation to permit
the ligation of a wider range of peptides, polypeptides, other
polymers and other molecules via an amide bond. The invention
further provides methods and uses for such proteins and derivatized
proteins.
BACKGROUND OF THE INVENTION
[0003] Over the past 30 years, medical attention has increasingly
turned to the possibility of using naturally produced proteins as
therapeutic drugs for the treatment of disease.
[0004] Recombinant DNA techniques have become the primary method
for commercial production of many polypeptides and proteins because
of the large quantities that can be produced in bacteria and other
host cells. Recombinant protein production involves transfecting or
transforming host cells with DNA encoding the desired exogenous
protein and growing the cells under conditions favoring expression
of the recombinant protein. E. coli and yeast are favored as hosts
because they can be made to produce recombinant proteins at high
titers (see, U.S. Pat. No. 5,756,672 (Builder et al.).
[0005] Numerous U.S. Patents have been issued with respect to
general bacterial expression of recombinant-DNA-encoded proteins
(see, for example, U.S. Pat. Nos. 4,565,785; 4,673,641; 4,738,921;
4,795,706; 4,710,473). Unfortunately, the use of recombinant DNA
techniques has not been universally successful. Under some
conditions, certain heterologous proteins expressed in large
quantities from bacterial hosts are precipitated within the cells
in dense aggregates, recognized as bright spots visible within the
enclosure of the cells under a phase-contrast microscope. These
aggregates of precipitated proteins are referred to as "refractile
bodies," and can constitute a significant portion of the total cell
protein (Brems et al., Biochemistry (1985) 24: 7662.
[0006] Recovery of protein from these bodies has presented numerous
problems, such as how to separate the protein encased within the
cell from the cellular material and proteins harboring it, and how
to recover the inclusion body protein in biologically active form.
For general review articles on refractile bodies, see Marston,
supra; Mitraki and King, Bio/Technology, 7: 690 (1989); Marston and
Hartley, Methods in Enzymol., 182: 264-276 (1990); Wetzel, "Protein
Aggregation In Vivo: Bacterial Inclusion Bodies and Mammalian
Amyloid," in Stability of Protein Pharmaceuticals: In Vivo Pathways
of Degradation and Strategies for Protein Stabilization, Ahem and
Manning (eds.) (Plenum Press, 1991); and Wetzel, "Enhanced Folding
and Stabilization of Proteins by Suppression of Aggregation In
Vitro and In Vivo," in Protein Engineering--A Practical Approach,
Rees, A. R. et al. (eds.) (IRL Press at Oxford University Press,
Oxford, 1991). A need therefore exists for an alternative way of
producing bioactive proteins.
[0007] One alternative to recombinant production of proteins
involves the use of the principles of organic chemistry to
synthesize proteins. Existing methods for the chemical synthesis of
proteins include stepwise solid phase synthesis, and fragment
condensation either in solution or on solid phase. The classic
stepwise solid phase synthesis of Merrifield involves covalently
linking an amino acid corresponding to the carboxy-terminal amino
acid of the desired peptide chain to a solid support and extending
the polypeptide chain toward the amino end by stepwise coupling of
activated amino acid derivatives having activated carboxyl groups.
After completion of the assembly of the fully protected solid phase
bound peptide chain, the peptide-solid phase covalent attachment is
cleaved by suitable chemistry and the protecting groups removed to
give the product polypeptide.
[0008] There are unfortunately multiple disadvantages to the
stepwise solid phase synthesis method, including the formation of
solid-phase bound by products that result from incomplete reaction
at the coupling and deprotection steps in each cycle. The longer
the peptide chain, the more challenging it is to obtain high-purity
well-defined products. The synthesis of proteins and large
polypeptides by this route is a time-consuming and laborious
task.
[0009] The solid phase fragment condensation approach (also known
as segment condensation) was designed to overcome the difficulties
in obtaining long polypeptides via the solid phase stepwise
synthesis method. The segment condensation method involves
preparation of several peptide segments by the solid phase stepwise
method, followed by cleavage from the solid phase and purification
of these maximally protected segments. The protected segments are
condensed one-by-one to the first segment, which is bound to the
solid phase. Often, however, technical difficulties are encountered
in many of the steps of solid phase segment condensation. See E.
Atherton, et al., "Solid Phase Fragment Condensation--The
Problems," in Innovation and Perspectives in Solid Phase Synthesis
11-25 (R. Epton, et al. 1990). For example, the use of protecting
groups on segments to block undesired ligating reactions can
frequently render the protected segments sparingly soluble,
interfering in efficient activation of the carboxyl group. Limited
solubility of protected segments also can interfere with
purification of protected segments. See K. Akaji et al., Chem.
Pharm. Bull.(Tokyo) 33:184-102 (1985). Protected segments are
difficult to characterize with respect to purity, covalent
structure, and are not amenable to high resolution analytical ESMS
(electrospray mass spectrometry) (based on charge). Racemization of
the C-terminal residue of each activated peptide segment is also a
problem, except if ligating is performed at Glycine residues.
Moreover, cleavage of the fully assembled, solid-phase bound
polypeptide from the solid phase and removal of the protecting
groups frequently can require harsh chemical procedures and long
reaction times that result in degradation of the fully assembled
polypeptide.
[0010] Segment condensation can be done in solution rather than on
solid phase. See H. Muramatsu et al., Biochem. and Biophys. Res.
Commn. 203(2):1131-1139 (1994). However, segment condensation in
solution requires purification of segments prior to ligation as
well as use of protecting groups on a range of different side chain
functional groups to prevent multiple undesired side reactions.
Moreover, the ligation in solution does not permit easy
purification and wash steps afforded by solid phase ligations.
Furthermore, the limitations with respect to solubility of
protected peptide segments and protected peptide intermediate
reaction products are exacerbated.
[0011] Chemical ligation of peptide segments has been explored in
order to overcome the solubility problems frequently encountered
with maximally protected peptide. Chemical ligation involves the
formation of a selective covalent linkage between a first chemical
component and a second chemical component. Unique, mutually
reactive, functional groups present on the first and second
components can be used to render the ligation reaction
chemoselective. For example, the chemical ligation of peptides and
polypeptides involves the chemoselective reaction of peptide or
polypeptide segments bearing compatible Unique, mutually-reactive,
C-terminal and N-terminal amino acid residues. Several different
chemistries have been utilized for this purpose, examples of which
include native chemical ligation (Dawson, et al., Science (1994)
266:776-779; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434),
oxime forming chemical ligation (Rose, et al., J. Amer. Chem. Soc.
(1994) 116:30-34), thioester forming ligation (Schnolzer, et al.,
Science (1992) 256:221-225), thioether forming ligation
(Englebretsen, et al., Tet. Leus. (1995) 36(48):8871-8874),
hydrazone forming ligation (Gaertner, et al., Bioconj. Chem. (1994)
5(4):333-338), and thiazolidine forming ligation and oxazolidine
forming ligation (Zhang, et al., Proc. Natl. Acad Sci. (1998)
95(16):9184-9189; Tam et al., WO 95/00846; U.S. Pat. No. 5,589,356)
or by other methods (Yan, L. Z. and Dawson, P. E., "Synthesis of
Peptides and Proteins without Cysteine Residues by Native Chemical
Ligation Combined with Desulfurization," J. Am. Chem. Soc. 2001,
123, 526-533, herein incorporated by reference; Gieselnan et al.,
Org. Lett. 2001 3(9):1331-1334; Saxon, E. et al., "Traceless"
Staudinger Ligation for the Chemoselective Synthesis of Amide
Bonds. Org. Lett. 2000, 2, 2141-2143).
[0012] Of these methods, only the native chemical ligation approach
yields a ligation product having a native amide (i.e. peptide) bond
at the ligation site. The original native chemical ligation
methodology (Dawson et al., supra; and WO 96/34878) has proven a
robust methodology for generating a native amide bond at the
ligation site. Native chemical ligation involves a chemoselective
reaction between a first peptide or polypeptide segment having a
C-terminal .alpha.-carboxythioester moiety and a second peptide or
polypeptide having an N-terminal cysteine residue. A thiol exchange
reaction yields an initial thioester-linked intermediate, which
spontaneously rearranges to give a native amide bond at the
ligation site while regenerating the cysteine side chain thiol. The
primary drawback of the original native chemical ligation approach
is that it requires an N-terminal cysteine, i.e., it only permits
the joining of peptides and polypeptide segments possessing an
N-terminal cysteine.
[0013] Notwithstanding this drawback, native chemical ligation of
peptides with N-terminal amino acids other than cysteine has been
reported (WO98/28434). In this approach, the ligation is performed
using a first peptide or polypeptide segment having a C-terminal
.alpha.-carboxythioester and a second peptide or polypeptide
segment having an N-terminal N-{thiol-substituted auxiliary} group
represented by the formula HS--CH.sub.2--CH.sub.2--O--NH-[peptide].
Following ligation, the N-{thiol substituted auxiliary} group is
removed by cleaving the HS--CH.sub.2--CH.sub.2--O-auxiliary group
to generate a native amide bond at the ligation site. One
limitation of this method is that the use of a mercaptoethoxy
auxiliary group can successfully lead to amide bond formation only
at a glycine residue. This produces a ligation product that upon
cleavage generates a glycine residue at the position of the
N-substituted amino acid of the second peptide or polypeptide
segment. As such, this embodiment of the method is only suitable if
one desires the ligation product of the reaction to contain a
glycine residue at this position, and in any event can be
problematic with respect to ligation yields, stability of
precursors, and the ability to remove the O-linked auxiliary group.
Although other auxiliary groups may be used, for example the
HSCH.sub.2CH.sub.2NH-[peptide], without limiting the reaction to
ligation at a glycine residue, such auxiliary groups cannot be
removed from the ligated product.
[0014] Accordingly, what is needed is a broadly applicable and
robust chemical ligation system that extends native chemical
ligation to a wide variety of different amino acid residues,
peptides, polypeptides, polymers and other molecules by means of an
effective, readily removable thiol-containing auxiliary group, and
that joins such molecules together with a native amide bond at the
ligation site. The present invention addresses these and other
needs.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 illustrates the overall reactions of pseudo native
chemical ligation.
[0016] FIG. 2 illustrates the overall process of preparing the
synthetic bioactive proteins of the present invention.
[0017] FIG. 3 depicts the basic structure of a preferred type of
synthetic erythropoiesis stimulating proteins.
[0018] FIG. 4 shows the general structure of circularly permuted
SEP analogs having no free amino or carboxy terminus. The pPEG
("precision PEG" as described herein) attachment sites are
positionally the same relative to the SEP analogs. The SEP analogs
are circularized by an oxime linkage between the new N-/C-terminal
positions, such as P126C and A125X. The total molecular weight of
the SEP analogs will range from about 50-80 kDa, depending on the
pPEG utilized for modification of a given analog, with the pPEG
mediated hydrodynamic MW estimated to be greater than about 100
kDa.
[0019] FIG. 5 shows the basic structure of synthetic bioactive GCSF
proteins. In the figure, "J" designates a non-naturally encoded
residue having a hydrophobic side chain.
[0020] FIG. 6 shows the structure of preferred synthetic RANTES
analogs. In the Figure, NNY=nonanoyl, X=non-naturally encoded amino
acid having a hydrophobic side chain, pPEG and FA=fatty acid
[0021] FIG. 7 depicts schematically the formation of branched-chain
pPEG polymer.
SUMMARY OF THE INVENTION
[0022] The present invention concerns methods and compositions for
extending the technique of native chemical ligation to permit the
ligation of a wider range of peptides, polypeptides, other polymers
and other molecules via an amide bond. The invention further
provides methods and uses for such proteins and derivatized
proteins. The invention is particularly suitable for use in the
synthesis of optionally polymer-modified, synthetic bioactive
proteins, and of pharmaceutical compositions that contain such
proteins.
[0023] In detail, the invention provides a synthetic protein
(especially a protein having a monomer molecular weight greater
than about 25 kDa) containing a pseudo-amino acid residue whose
side chain has the formula: --S--R.sub.aa where R.sub.aa is an
optionally substituted terminal portion of a ribosomally-specified
amino acid side chain, or an analog of the terminal portion of a
ribosomally-specified amino acid side chain.
[0024] The invention particularly provides the embodiment of such a
synthetic protein wherein the protein has a biological activity
possessed by a ribosomally-produced bioactive protein.
[0025] The invention further concerns such synthetic proteins
wherein a plurality of amino acid residues of the protein are bound
to adjacent amino acid residues by an amide bond, and such
synthetic proteins wherein a plurality of amino acid residues
wherein at least one of such amino acid residues is bonded to an
adjacent amino acid residue by a non-amide bond (e.g., a thioester
bond, a thioether bond, and an oxime bond).
[0026] The invention further concerns such synthetic proteins
wherein the pseudo-amino acid residue is a D-configuration amino
acid residue or is an an L-configuration amino acid residue, or
wherein such synthetic proteins contain both pseudo-amino acid
residue(s) in the D-configuration and pseud-amino acid residue(s)
in the L-configuration.
[0027] The invention further concerns such synthetic proteins
wherein the pseudo-amino residue is selected from the group
consisting of a pseudo-arginine; a pseudo-asparagine; a
pseudo-aspartate; a pseudo-dopamine; a pseudo-glutamate; a
pseudo-glutamine; a pseudo-histidine; a pseudo-isoleucine; a
pseudo-leucine; a pseudo-lysine; a pseudo-methionine; a
pseudo-phenyalanine; a pseudo-serine; a pseudo-threonine; a
pseudo-tryptophan; a pseudo-tyrosine; and a pseudo-valine.
[0028] The invention further concerns such synthetic proteins
wherein the ribosomally-specified bioactive protein is a mammalian
protein (e.g., a human, simian, bovine, murine, porcine, ovine, or
equine protein).
[0029] The invention further concerns such synthetic proteins
wherein the protein has a bioactivity selected of a protein
receptor or fragment thereof, of a protein receptor ligand or
fragment thereof, or of a cytokine (especially wherein the cytokine
is selected from the group consisting of an interleukin, a
lymphokine, a RANTES protein, an erythropoiesis stimulating
protein, tumor necrosis factor (TNF), an interferon, a growth
factors and a single peptide hormone).
[0030] The invention further concerns the synthetic proteins SEP-0,
SEP-1, and SEP-3.
[0031] The invention further concerns all such synthetic proteins
wherein one or more of their amino acid residues are modified by
one or more polymer adducts, and particularly wherein the synthetic
protein comprises one or more amino acid residues that are modified
by one or more polymer adducts at amino acid residue(s) that
correspond to at least one of the glycosylation sites of a
ribosomally-encoded bioactive protein.
[0032] The invention also provides a molecularly homogeneous
pharmaceutical composition comprising a synthetic protein, wherein
the protein possesses a biological activity that mimics a
biological activity associated with a ribosomally-specified
bioactive mammalian protein, and has a monomer molecular weight
greater than about 25 kDa, and contains a pseudo-amino acid residue
whose side chain has the formula: --S--R.sub.aa, where R.sub.aa is
selected from the group consisting of an optionally substituted
terminal portion of a ribosomally-specified amino acid side chain,
or an analog thereof.
[0033] The invention also provides a method of treating a human
disease or condition that comprises administering to an individual
in need of such treatment an effective amount of a pharmaceutical
composition comprising one or more molecularly homogeneous
pharmaceutical compositions each comprising a synthetic protein,
wherein the synthetic protein has a monomer molecular weight
greater than about 25 kDa, and contains a pseudo-amino acid residue
whose side chain has the formula: --S--R.sub.aa, where R.sub.aa is
selected from the group consisting of an optionally substituted
terminal portion of a ribosomally-specified amino acid side chain,
or an analog thereof; the synthetic protein possessing a biological
activity that mimics a biological activity of a
ribosomally-specified bioactive human protein receptor or fragment
thereof, protein receptor ligand or fragment thereof, or a
cytokine.
[0034] The invention also provides a method for synthesizing a
desired polypeptide, and the desired polypeptide made through such
method, wherein the desired polypeptide has the formula:
aa.sub.NH.sub.2-Q-aa.sub.x-aa.sub.y-W-aa.sub.COOH where Q and W
each denote the optional presence of one or more additional amino
acid residues, aa.sub.NH.sub.2 denotes the N-terminal amino acid
residue of the polypeptide; aa.sub.x and aa.sub.y denote internal
adjacent amino acid residues, having side chains x and y,
respectively, and aa.sub.COOH denotes the C-terminal amino acid
residue of the polypeptide; the method comprising: [0035] (A)
ligating a first peptide having the formula:
aa.sub.NH.sub.2-Q-aa.sub.x-COSR, wherein R is any group compatible
with the thioester group, including, but not limited to, aryl,
benzyl, and alkyl groups, to a second peptide having the formula:
Cys-W-aa.sub.COOH to thereby form the polypeptide:
aa.sub.NH.sub.2-Q-aa.sub.x-Cys-W-aa.sub.COOH; and [0036] (B)
incubating the polypeptide in the presence of a reagent
R.sub.aa--X, where R.sub.aa is a group whose structure mimics the
terminal portion of the side chain of amino acid residue aa.sub.y;
and X is a good leaving group (especially a halogen, such as F, I,
or Br); the incubation being under conditions sufficient to form
the desired polypeptide.
[0037] The invention further provides the embodiment of such method
and polypeptide wherein the amino acid residue aa.sub.y is selected
from the group consisting of a pseudo-arginine; a
pseudo-asparagine; a pseudo-aspartate; a pseudo-dopamine; a
pseudo-glutamate; a pseudo-glutamine; a pseudo-histidine; a
pseudo-isoleucine; a pseudo-leucine; a pseudo-lysine; a
pseudo-methionine; a pseudo-phenyalanine; a pseudo-serine; a
pseudo-threonine; a pseudo-tryptophan; a pseudo-tyrosine; and a
pseudo-valine.
DESCRIPTION OF THE PREFERRED EMBODIMENT
[0038] The present invention concerns methods and compositions for
extending the technique of native chemical ligation to permit the
ligation of a wider range of peptides, polypeptides, other polymers
and other molecules via an amide bond, thus facilitating the
chemical synthesis of such molecules. The invention further
provides methods and uses for such proteins and derivatized
proteins. The invention is particularly suitable for use in the
synthesis of optionally polymer-modified, synthetic bioactive
proteins, preferably having a monomer molecular weight of greater
than about 25 kDa, and of pharmaceutical compositions that contain
such proteins.
I. The Chemical Synthesis of Bioactive Peptides and Proteins
[0039] Typically, the synthesis of bioactive peptides and proteins
employs the "Merrifield"-chemistry stepwise solid phase peptide
synthesis protocol developed in the early 1960's, using standard
automated peptide synthesizers. Such synthesis may employ solid or
solution phase ligation strategies. While such chemistry may be
readily employed to produce many polypeptides, it is unsuitable for
the production of proteins or large polypeptides due to associated
yield losses, by-product production, and incomplete reactions. To
address these limitations, techniques of chemical ligation have
been developed that permit one to ligate together preformed peptide
fragments in order to achieve the synthesis of larger polypeptides
and proteins.
[0040] Chemical ligation involves the formation of a selective
covalent linkage between a first chemical component and a second
chemical component. Unique, mutually reactive, functional groups
present on the first and second components can be used to render
the ligation reaction chemoselective. For example, the chemical
ligation of peptides and polypeptides involves the chemoselective
reaction of peptide or polypeptide segments bearing compatible
Unique, mutually reactive, C-terminal and N-terminal amino acid
residues. Several different chemistries have been utilized for this
purpose, examples of which include native chemical ligation
(Dawson, et al., Science (1994) 266:776-779; Kent, et al., WO
96/34878; Kent, et al., WO 98/28434), oxime forming chemical
ligation (Rose, et al., J. Amer. Chem. Soc. (1994) 116:30-34),
thioester forming ligation (Schnolzer, et al., Science (1992)
256:221-225), thioether forming ligation (Englebretsen, et al.,
Tet. Letts. (1995) 36(48):8871-8874), hydrazone forming ligation
(Gaertner, et al., Bioconj. Chem. (1994) 5(4):333-338), and
thiazolidine forming ligation and oxazolidine forming ligation
(Zhang, et al., Proc. Natl. Acad. Sci. (1998) 95(16):9184-9189;
Tam, et al., WO 95/00846; U.S. Pat. No. 5,589,356) or by other
methods (Yan, L. Z. and Dawson, P. E., "Synthesis of Peptides and
Proteins without Cysteine Residues by Native Chemical Ligation
Combined with Desulfurization," J. Am. Chem. Soc. 2001, 123,
526-533, herein incorporated by reference; Gieselnan et al., Org.
Lett. 2001 3(9):1331-1334; Saxon, E. et al., "Traceless" Staudinger
Ligation for the Chemoselective Synthesis of Amide Bonds. Org.
Lett. 2000, 2, 2141-2143).
[0041] Where the ligation involves the joining of a polypeptide
that possesses an N-terminal cysteine residue, the procedure of
native chemical ligation is preferably employed (Dawson, et al.,
Science (1994) 266:776-779; Kent, et al., WO 96/34878; Kent, et
al., WO 98/28434; U.S. patent application Ser. No. 09/097,094, all
herein incorporated by reference). Native chemical ligation
involves a chemoselective reaction between a first peptide or
polypeptide segment having a C-terminal .alpha.-carboxythioester
moiety and a second peptide or polypeptide having an N-terminal
cysteine residue. A thiol exchange reaction yields an initial
thioester-linked intermediate, which spontaneously rearranges to
give a native amide bond at the ligation site while regenerating
the cysteine side chain thiol. In many instances, the sequence of
the natural protein will comprise suitably placed cysteine residues
such that polypeptide fragments having an N-terminal cysteine
residue may be synthesized and used in a native chemical ligation
reaction.
[0042] Although this methodology has proven to be practical, robust
and useful for the synthesis of large polypeptides and proteins,
its requirements for a cysteine residue at the site of ligation
limits its applicability. Cysteine is one of the least common amino
acids. A typical protein domain comprises 150-200 amino acids; many
proteins contain regions of greater than 60 amino acids in length
that contain no cysteine residues. One solution to this problem
involves designing a non-naturally occurring protein in which one
or more of the protein's naturally occurring amino acid residues
have been replaced with cysteine residues, thus permitting the
native chemical ligation method to be employed. This solution is
often complicated by the unpredictable effects that the
introduction of a cysteine residue may have on protein structure or
function. Such cysteine residues may be used to covalently
immobilize the synthesized protein.
[0043] The present invention provides two alternative solutions to
this problem that may be employed separately or conjunctively. The
first such solution (termed herein "Pseudo-Native Chemical
Ligation") involves the use of non-naturally occurring pseudo-amino
acid residues at preselected positions in the peptides employed in
the protein synthesis. The structures of such pseudo-amino acids
mimic both the structures of cysteine and the structures of the
amino acids that are naturally found at such preselected positions
in the protein being synthesized. The second such solution (termed
herein "Extended Native Chemical Ligation") is described in U.S.
Patent Application Ser. No. 60/231,339 (Kent, et al., filed: Sep.
8, 2000), herein incorporated by reference, and involves ligating a
first component comprising a carboxyl thioester, and more
preferably, an .alpha.-carboxyl thioester with a second component
comprising an acid stable N-substituted, and preferably,
Na-substituted, 2 or 3 carbon chain amino alkyl or aryl thiol. Each
of these solutions is discussed in detail below.
[0044] A. Pseudo-Native Chemical Ligation
[0045] Pseudo-native chemical ligation is directed to the
thioalkylation of cysteine side chains generated at ligation sites
from native chemical ligation. A preferred aspect is thioalkylation
of cysteine ligation sites wherein at least one peptide contains a
native cysteine having its thiol side chain protected with a
suitable protecting group.
[0046] In a preferred embodiment of the invention, the thiol moiety
of a cysteine group is modified into a desired side chain, for
example, into the side chain of a ribosomally-specified amino acid,
an analog of such an amino acid, or into a
non-ribosomally-specified amino acid. As used herein, a
ribosomally-specified amino acid is an amino acid that is
recognized by ribosomes in the process of protein translation and
can be incorporated into a ribosomally produced protein.
Considerable published literature exists describing chemical
modifications of the cysteine side chain thiol moiety (see, e.g.,
"Current Protocols in Protein Science," Edited by: John E. Coligan
et al., John Wiley & Sons, NY (2000)). Kaiser, E. T. has
described the conversion of cysteine residue side chains to mimic
the chemical properties of a naturally occurring amino acid side
chain (see, e.g., Kaiser, E. T. et al., "Chemical Mutation Of
Enzyme Active Sites," Science. Nov. 2, 1984;226(4674):505-11).
Additionally, the use of a cysteine side chain to introduce a label
into a peptide or protein has been described. Cysteine side chain
modifications are reviewed in Chemistry of Protein Conjugation and
Crosslinking, S. S. Wong, (1991, CRC Press); Chemical Modification
of Proteins, Gary E. Means et al., (1971, Holden-Day), Chemical
Modification of Proteins: Selected methods and analytical
procedures, Glazer, A. N. et al. (1975, Elsevier); Chemical
Reagents for Protein Modification, R L Lundblad (1991, CRC Press).
Tam et al. (Biopolymers (1998) 46:319-327) have disclosed the use
of homocysteine (--CH.sub.2--CH.sub.2--SH) for non-cys native
chemical ligation, followed by thioalkylation using methyl
p-nitrobenzenesulfonate (methylating reagent) to convert the
homocysteine side chain to a native methionine side chain
(--CH.sub.2--CH.sub.2--S--CH.sub.3). The present invention also can
be used for converting homocysteines to pseudo amino acids as well,
i.e., to amino acids other than methionine. However, as with the
conversion of cysteines described herein, in accordance with the
present invention it is necessary to use protecting groups to avoid
destruction of native cysteines involved in disulfide pairing for
peptides that contain at least one native cysteine that one does
not wish to convert. Suitable protecting groups are described
below.
[0047] While the method of pseudo-native chemical ligation does not
facilitate the mimicking of the side chains of certain
ribosomally-specified amino acids (e.g., the side chains of
glycine, alanine, valine, and proline) (alanine's side chain can,
however, be formed through a desulfurization reaction (Yan, L. Z.
and Dawson, P. E., "Synthesis of Peptides and Proteins without
Cysteine Residues by Native Chemical Ligation Combined with
Desulfurization," J. Am. Chem. Soc. 2001, 123, 526-533, herein
incorporated by reference), it may be used to form side chains that
mimic many ribosomally-specified or non-encoded amino acids. Amino
acids produced in accordance with the pseudo-native chemical
ligation method of the present invention will contain a thioether
linkage, and will have no beta-branching (in that they will all
include a methyl group at the beta position, i.e.,
aa-.beta.CH.sub.2--S--. Thus, the pseudo-amino acid versions of the
beta-branched amino acids, isoleucine and threonine can be made to
have the pendant side chain structure, without having the beta
geometry and its attendant constraints.
[0048] Significantly, the methods of the present invention may be
used to form amino acid side chains that are the same length as
that of ribosomally-specified amino acids, or are longer or shorter
than such length. Such alteration in side chain length can be used
to stabilize (or destabilize) the three-dimensional conformation to
increase protein stability (or to enhance the ability of the
protein to alter its conformation and thereby accept a different
range of substrates, inhibitors, receptors, ligands, etc. relative
to those accepted by the naturally occurring protein. For example,
Cys-CH.sub.2--SH+Br--CH.sub.2--COOH yields
Cys-CH.sub.2--S--CH.sub.2--COOH (such "pseudo-glutamic acid" has
one additional side chain atom, namely the --S-- group;
alternatively, if used in the place of aspartic acid, it will
possess two additional side chain atoms, namely a --CH.sub.2--S--
group). Other side chains have the same number of atoms in the side
chain, but differ by inclusion of the thioether linkage (--S--).
For example, Cys-CH.sub.2--SH+Br--CH.sub.2--CH.sub.2--NH--PG,
followed by removal of PG yields
Cys-CH.sub.2--S--CH.sub.2--CH.sub.2--NH.sub.2. The resulting
structure has no additional atoms in the side chain, but one
--CH.sub.2-group is replaced with --S--. Methionine is another
example here, Cys-CH.sub.2--SH+I--CH.sub.2--CH.sub.3 yields
Cys-CH.sub.2--S--CH.sub.2--CH.sub.3 (versus native met structure of
Met-CH.sub.2--CH.sub.2--S--CH.sub.3); thus the thioether is
relocated. Arginine also:
Cys-CH.sub.2--SH+B--CH.sub.2--NH--CH((--NH.sub.2)(.dbd.NH.sub.2.sup.+))
yields
Cys-CH.sub.2--S--CH.sub.2--NH--CH((--NH.sub.2)(.dbd.NH.sub.2.sup.+-
)). Preferably, protection of reactive amino groups, particularly
for the constructing pseudo lysine can be employed to avoid
unwanted side reactions. Once the thioalkylation reaction is
performed, the protecting group can be removed.
[0049] In general, where the desire is to mimic a naturally
occurring protein as closely as possible, it is most preferred to
employ a pseudo amino acid molecule having a side chain length that
is the same length as that of the ribosomally-specified amino acid
normally present at such position in the protein; it is less
preferred to employ a pseudo amino acid molecule having a side
chain length that is one atom longer than that of the
ribosomally-specified amino acid, and still less preferred to
employ a pseudo amino acid molecule having a side chain length that
is two atoms longer than that of the ribosomally-specified amino
acid. Moreover, its is preferred to select a cysteine ligation site
that is in a location where genetic changes are not likely to
disrupt function or where amino acids at that site in related
proteins are conserved. Such sites can be identified by alanine
scanning, homology modeling, and other methods.
[0050] In pseudo-native chemical ligation, a peptide containing an
amino terminal cysteine residue is ligated to a peptide having a
carboxy terminal thioester, as in native chemical ligation. The
thiol side chain of the cysteine is then reacted with a compound of
the formula R.sub.aa--X, where X is a good leaving group, and
R.sub.aa is a group whose structure mimics the terminal portion of
the side chain of an ribosomally-specified or synthetic amino
acid.
[0051] Significantly, the reactions of pseudo-native chemical
ligation work with either the natural L-configuration of cysteine
side chain, or the D-configuration. Use of the D-configuration can
impart protease resistance at that ligation site, and thus may be
desired when increased stability to proteolysis is desired.
However, in using the D-cysteine, the backbone structure at that
side will be altered. Such alteration, in addition to protease
resistance, may be desired to alter bioactivity. However, to
minimize impact on bioactivity, it is preferred to locate the
D-cysteine at a site of high flexibility, such as a disorded
region, such as at a disorded loop that will be located on the
surface of the resulting folded molecule, on at a disorded terminus
of the molecule. Desirably, the reactions of pseudo-native chemical
ligation may be used to place large, charged side chains (e.g., the
side chains of Lys, Arg, Asp or Glu) on the surface of the
synthesized molecule.
[0052] Examples of suitable good leaving groups, X, include
halogens, especially Iodine and Bromine. Examples of R.sub.aa
groups include PO.sub.4, COOH, COO, CONH.sub.2, guanidinium, amine,
alkyl, substituted alkyl, aryl, substituted aryl, imidazole,
alkylated imidazole, indole, or alkylated indole groups.
[0053] The selection of which R.sub.aa to employ will depend upon
the amino acid side chain desired to be present at a particular
position. Thus, for example, a desired polypeptide or protein
having the amino acid sequence:
aa.sub.NH.sub.2-Q-aa.sub.x-aa.sub.y-W-aa.sub.COOH
[0054] where Q and W each denote the optional presence or absence
of additional amino acid residues, and aa.sub.x and aa.sub.y denote
internal adjacent residues (having side chains x and y,
respectively), and aa.sub.NH.sub.2 and respectively denote the
amino (N--) terminal residue and the carboxy (C--) terminal residue
of the polypeptide or protein an be synthesized by preparing two
peptide fragments: aa.sub.NH.sub.2-Q-aa.sub.x-COSR and
Cys-W-aa.sub.COOH
[0055] where Cys denote the replacement of aa.sub.y with cysteine,
and R is any group compatible with the thioester group, including,
but not limited to, aryl, benzyl, and alkyl groups. Examples of R
include 3-carboxy-4-nitrophenyl thioesters, benzyl esters, and
mercaptoproprionic acid leucine esters (See, e.g., Dawson et al.,
Science (1994) 266:776-779; Canne et al. Tetrahedron Lett. (1995)
36:1217-1220; Kent, et al., WO 96/34878; Kent, et al., WO 98/28434;
Ingenito et al., JACS (1999) 121(49):11369-11374; and Hackeng et
al., Proc. Natl. Acad. Sci. U.S.A. (1999) 96:10068-10073). Other
examples include dithiothreitol, or alkyl or aryl thioesters, which
can be produced by intein-mediated biological techniques, which
also are well known (See, e.g., Chong et al., Gene (1997)
192:277-281; Chong et al., Nucl. Acids Res. (1998) 26:5109-5115;
Evans et al., Protein Science (1998) 7:2256-2264; and Cotton et
al., Chemistry & Biology (1999) 6(9):247-256); and then
ligating the fragments together to form:
aa.sub.NH.sub.2-Q-aa.sub.x-Cys-W-aa.sub.COOH--
[0056] The ligated fragment is then reacted with R.sub.y--X, where
R.sub.y is a side group that mimics the structure of the y side
chain). The reaction is conducted under conditions sufficient to
convert the thiol group of the cysteine into a "pseudo-y" side
chain. For example, if R.sub.aa is selected to be CH.sub.2--COOH or
(CH.sub.2).sub.2--COOH, then the reaction will lead to the
formation an amino acid residue that mimics the structure and
function of aspartic acid ("pseudo-Asp") or glutamic acid
("pseudo-Glu"). As will be appreciated, in light of the above
description, more complicated synthesis can be conducted employing
more than two peptide fragments.
[0057] Native chemical ligation involving "pseudo amino acids"
according to the present invention significantly expands the
applicability of the native chemical ligation method for the
chemical synthesis of proteins. Native chemical ligation involving
"pseudo amino acids" uses the same chemistry in the ligation step,
forming a peptide bond at the Cys residue with a native
thiol-containing side chain functionality; in a unique second step,
the native thiol-containing side chain of the Cys at the site of
ligation is converted by chemoselective reaction to yield a
non-native side chain that contains the same functional group as a
genetically-encoded amino acid, but a non-native side chain.
[0058] For example, the cysteine at the ligation site can be
reacted with bromoacetic acid (or other haloacetic acid) at neutral
pH in water; only the unprotected thiol of the Cys at the ligation
site reacts under these conditions to give a polypeptide containing
an S-carboxymethylated residue at the ligation site, viz:
[0059] This non-native amino ##STR1## acid contains a carboxyl
moiety on the side chain, the same functional group as a glutamic
acid or an aspartic acid residue. The S-carboxymethylated Cys is
referred to herein as a "pseudo glutamic acid" ("pseudo-glutamate,"
"pseudo-Glu") (where one extra side chain atom is present and is
the --S-- group forming a thioether linkage) (it also may be
referred to as a "pseudo-aspartic acid" where two extra side chain
atoms are present, one of which is the --S-- group forming a
thioether linkage). For many proteins, the simple preservation of
the charged carboxylate side chain even in this "pseudo amino acid"
will be sufficient to retain or impart desired properties to the
protein. A key difference is that it functions as a native chemical
ligation site at a position devoid of a suitable cysteine.
[0060] Use of other reagents to modify the side chain thiol in Cys
residues at the ligation site(s) can give other "pseudo amino acid"
residues. For example, reaction of the thiol with a haloacetamide,
e.g. bromoacetamide, will give a "pseudo glutamine" at the ligation
site, viz: ##STR2## Similarly, reaction with an N-protected
haloalkylamine, e.g. ##STR3## followed by removal of the amino
protecting group will give a `pseudo-lysine` residue, viz: ##STR4##
Reaction of the Cys residue(s) at the ligation site(s) with a
suitable haloalkane, e.g. ##STR5## will yield a `pseudo leucine`
amino acid, viz: ##STR6## Other suitably chosen aryl-containing
halides, where R can be H, OH, aryl etc., e.g. ##STR7## can be used
similarly to generate pseudo amino acids corresponding to Phe, Tyr,
or Trp residues, viz:
[0061] A significant ##STR8## feature of this approach is that
cysteine residues that one does not wish to modify in the reacting
segments be side chain protected, e.g. as Cys(Acm), to prevent
chemical modification of Cys residues other than the one(s) at the
ligation site(s), or that any other Cys residues in the reacting
segments be intended for the simultaneous formation of identical
`pseudo amino acids` by the chemical modification reaction after
ligation. It will be appreciated that the same principles apply to
the use of homocysteine at the ligation site. It will also be
appreciated that selenocysteine can be employed at a ligation site
through selenocysteine-mediated native chemical ligation, and
converted by similar alkylation reactions to generate pseudo amino
acids of the invention, where a selenoether group replaces the
thioether group.
[0062] As used herein, the symbol (p denotes a benzyl group; IM
denotes an imidazole group, and IV denotes an indole group; PG
denotes a protecting group. Below is a summary of R.sub.aa side
groups that may be used to synthesize peptides containing
pseudo-amino acid residues in accordance with the present invention
(where X is a halogen (I, Br, Cl, F, with I and Br preferred for
most, and F preferred for .phi. attachment):
Basic Amino Acids:
Lys (No Extra Atoms) --CH.sub.2--SH+X--CH.sub.2--CH.sub.2--NH--PG,
followed by deprotection gives
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH.sub.2 Arg (No Extra Atoms)
--CH.sub.2--SH+X--CH.sub.2--NH--C((NH.sub.2)(.dbd.NH.sub.2+)) gives
--CH.sub.2--S--CH.sub.2--NH--C((NH.sub.2)(.dbd.NH.sub.2+)) His (2
Extra Atoms) --CH.sub.2--SH+X--CH.sub.2--IM-PG, followed by
deprotection gives --CH.sub.2--S--CH.sub.2--IM Acidic Amino Acids:
Asp (2 Extra Atoms) --CH.sub.2--SH+X--CH.sub.2--COOH gives
--CH.sub.2--S--CH.sub.2--COOH Glu (1 Extra Atoms)
--CH.sub.2--SH+X--CH.sub.2--COOH gives
--CH.sub.2--S--CH.sub.2--COOH Uncharged Polar Amino Acids: Tyr (No
or 1 Extra Atom) --CH.sub.2--SH+F-.phi.-pOH gives
--CH.sub.2--S-.phi.-pOH)(no extra atoms, same geometry
--CH.sub.2--SH+Br/I--CH.sub.2-.phi.-pOH gives
--CH.sub.2--S--CH.sub.2-.phi.-pOH (1 extra atom) Gln (1 Extra Atom)
--CH.sub.2--SH+X--CH.sub.2--C(O)(NH.sub.2) gives
--CH.sub.2--S--CH.sub.2--C(O)(NH.sub.2) Asn (2 Extra Atoms)
--CH.sub.2--SH+X--CH.sub.2--C(O)(NH.sub.2) gives
--CH.sub.2--S--CH.sub.2--C(O)(NH.sub.2) Ser (2 or 3 Extra Atoms)
--CH.sub.2--SH+X--CH.sub.2OH) gives --CH.sub.2--S--CH.sub.2OH (2
extra atoms) --CH.sub.2--SH+X--CH.sub.2--CH.sub.2OH) gives
--CH.sub.2--S--CH.sub.2--CH.sub.2OH (3 extra atoms) Thr (2 or 3
Extra Atoms, Missing Beta Branching)
--CH.sub.2--SH+X--CH((CH.sub.3)(O--PG)) followed by removal of PG
gives --CH.sub.2--S--CH((CH.sub.3)(OH)) (2 extra atom)
--CH.sub.2--SH+X--CH.sub.2--CH((CH.sub.3)(O--PG)) followed by
removal of PG gives --CH.sub.2--S--CH.sub.2--CH((CH.sub.3)(OH)) (3
extra atoms) Non-Polar Amino Acids: Leu (1 or 2 Extra Atoms)
--CH.sub.2--SH+X--CH((CH.sub.3)(CH.sub.3)) gives
--CH.sub.2--S--CH((CH.sub.3)(CH.sub.3)) (1 extra atom)
--CH.sub.2--SH+X--CH.sub.2--CH((CH.sub.3)(CH.sub.3)) gives
--CH.sub.2--S--CH.sub.2--CH((CH.sub.3)(CH.sub.3)) (2 extra atoms)
Ile (2 or 3 Extra Atoms, Missing Deta Branching)
--CH.sub.2--SH+X--CH(CH.sub.3)--CH.sub.2--CH.sub.3 gives
--CH.sub.2--S--CH(CH.sub.3)--CH.sub.2--CH.sub.3 (2 extra atoms)
--CH.sub.2--SH+X--CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.3 gives
--CH.sub.2--S--CH.sub.2--CH(CH.sub.3)--CH.sub.2--CH.sub.3 (3 extra
atoms) Phe (1 or 2 Extra Atoms) --CH.sub.2--SH+F-.phi. gives
--CH.sub.2--S-.phi. (1 extra atom)
--CH.sub.2--SH+Br/I--CH.sub.2-.phi. gives
--CH.sub.2--S--CH.sub.2-.phi. (2 extra atoms) Met (No Extra Atoms)
--CH.sub.2--SH+I--CH.sub.2--CH.sub.3 gives
--CH.sub.2--S--CH.sub.2--CH.sub.3 Trp (1 Extra Atom)
--CH.sub.2--SH+F--IN gives --CH.sub.2--S--IN
[0063] The general pseudo chemical ligation reaction is illustrated
in FIG. 1. Preferred R.sub.aa and X groups that can be used to form
preferred pseudo-amino acids are shown in Table I. Residues are
shown as uncharged, however, it will be appreciated that ionizable
groups such as NH.sub.2 or COOH may be in their charged form, or
provided as salts. All reactions except that used to form
pseudo-lysine are aqueous and are conducted at a pH of 8-9, where
the stocheometry and optimization of the reactions can be adjusted
following standard protocols. Pseudo-lysine is formed in an aqueous
reaction conducted at pH 8-9, followed by reaction in
piperidine/H.sub.2O (trifluoracetic acid (TFA)). TABLE-US-00001
TABLE I Mimics Pseudo-Amino or R.sub.aa -X Reagent Product of
Reaction Acid Replaces X--CH.sub.2COOH (X = I, Br) ##STR9##
Pseudo-Asp (+2 atoms) Pseudo-Glu (+1 atoms) Aspartic Acid Glutamic
Acid X(CH.sub.2).sub.2COOH (X = I, Br) ##STR10## Pseudo-Glu (+2
atoms) Glutamic Acid X--(CH.sub.2)----C(O)NH.sub.2(X = I, Br)
##STR11## Pseudo-Gln (+1 atom) Pseudo-Asn (+2 atoms) Glutamine
Asparagine X--(CH.sub.2OH (X = I, Br) ##STR12## Pseudo-Ser (+2
atoms) Serine X--CHOHCH.sub.3(X = I, Br) ##STR13## Pseudo-Thr (+2
atoms) Threonine X--CH.sub.2CHOHCH.sub.3(X = I, Br) ##STR14##
Pseudo-Thr (+3 atoms) Threonine X--CH.sub.2CH.sub.2OH (X = I, Br)
##STR15## Pseudo-Ser (+3 atoms) Serine X--(CH.sub.2).sub.2NH--PG (X
= I, Br) ##STR16## Pseudo-Lys Lysine
X--CH.sub.2NH----C(NH.sub.2).sub.2(X = I, Br) ##STR17## Pseudo-Arg
Arginine X--(CH.sub.2).sub.2NH----C(NH.sub.2).sub.2(X = I, Br)
##STR18## Pseudo-Arg (+1 atom) Arginine X--CH.sub.2CH.sub.3(X = I,
Br) ##STR19## Pseudo-Met Methionine X--.PHI.(X = F) ##STR20##
Pseudo-Phe (+1 atoms) Phenyl- alanine X--CH.sub.2.PHI.(X = I, Br)
##STR21## Pseudo-Phe (+2 atoms) Phenyl- alanine X--.PHI.--OH para
(X = I, Br) ##STR22## Pseudo-Tyr Tyrosine X--CH.sub.2.PHI.--0H
(para) (X = I, Br) ##STR23## Pseudo-Tyr (+1 atom) Tyrosine
X--CH.sub.2.PHI.--OPO.sub.3(para) (X = I, Br) ##STR24## Pseudo-
Phospho-tyr Phospho- tyrosine X--CH.sub.2.PHI.--(OH).sub.2(meta
para) (X = I, Br) ##STR25## Pseudo-Dopa Dopamine
X--CH(COOH).sub.2(X = I, Br) ##STR26## Pseudo-Gla .gamma.carboxy
Glutamic Acid X--CH.sub.2--IM--PG (X = I, Br) ##STR27## Pseudo-His
Histidine X--CH.sub.2--IN (X = F,I, Br) ##STR28## Pseudo-Trp
Tryptophan X--CH----(CH.sub.3).sub.2(X = I, Br) ##STR29##
Pseudo-Leu (+1 atoms) Pseudo-Val (+2 atoms) Leucine Valine
X--CH.sub.2CH----(CH.sub.3).sub.2(X = I, Br) ##STR30## Pseudo-Leu
(+2 atoms) Leucine X--CH(CH)----CH.sub.2--CH.sub.3(X = I, Br)
##STR31## Pseudo-Ile (+2 atoms) Isoleucine
X--CH.sub.2----CH(CH.sub.3)----CH.sub.2--CH.sub.3(X = I, Br)
##STR32## Pseudo-Ile (+3 atoms) Isoleucine
X--(CH.sub.2).sub.2NH----C(O)CF.sub.3(X = I, Br) ##STR33##
Pseudo-N- (ethyl) trifluoroacet- amide N-(ethyl) trifluroacet-
amide X-ammonium 4- chloro-7- sulfobenzo- furazan (X-SBF) (X = I,
Br) ##STR34## Pseudo-SBF Ammonium 4-chloro-7- sulfobenzo- furazan
(SBF) (Pierce Chemi cal Company) X--CH.sub.2--ammonium 4- chloro-7-
sulfobenzo- furazan (X-SBF) (X = I, Br) ##STR35## Pseudo methyl-SBF
Ammonium 4-chloro-7- sulfobenzo- furazan (SBF) (Pierce Chemical
Company)
[0064] In a further embodiment of the present invention, the thiol
group of the cysteine residue can be oxidized
(--SH.fwdarw.--SO.sub.3.sup.-), or replaced with oxygen to form
serine (--SH.fwdarw.--OH). Such replacement can be accomplished by
reacting the cysteine with tosyl-Cl (Tos-Cl) thereby converting the
thiol group, --SH, into an --S-Tos group. In the presence of strong
base, OH replaces the Tos substituent, thereby forming the desired
--OH group.
[0065] Additionally, the thiol group of the cysteine can be
modified through a Michael addition reaction, as shown below:
##STR36## where R is H, alky, aryl, alkylaryl, amino, or is a
polymer molecule, which may be brancehed or unbranched, random or
block.
[0066] A further aspect of the present invention is directed to the
use of a peptide fragment whose amino terminal cysteine residue has
been modified to contain an alkylamino (and preferably a
methylamino) moiety: ##STR37##
[0067] By protecting the amino group of the alkylamino moiety, one
can ligate the PeptideA to a second peptide having the general
formula: Peptide B-.alpha.COSR. Upon removal of the protecting
group, an additional Peptide having the general formula: Peptide
C-.alpha.COSR can be ligated to the previously ligated peptides, to
form a branched peptide complex: ##STR38##
[0068] Since the branched protein still contains the thiol group of
the cysteine, it will react with an R.sub.aa--X molecule in the
manner described above, to thereby permit further modifications of
the side chain. In particular, by using an alkylamide as the R
substituent, an additional amino group can be introduced into the
molecule, thereby permitting further branching, or further ligation
reactions: ##STR39##
[0069] In a further embodiment of the present invention, the
modification of the cysteine side chain can be used to introduce
any of a variety of labels or binding molecules. For example,
labels detectable by electron spin resonance, fluorescence (e.g.,
ammonium 4-chloro-7-sulfobenzo-furazan (SBF) Pierce Chemical Co.)or
magnetic imaging may be employed. Such labeling is useful in the
diagnosis of diseases and conditions, as well as in the analysis of
molecular interactions (e.g., for measuring distances in integral
membrane proteins). The methodology of the present invention has
several salient advantages with respect to the use of labels and
binding molecules. Such substituents can be incorporated in a site
specific, non-random manner. Additionally, the label moiety need
only be stable to subsequent chemical ligation steps (if any). Such
reactions are generally conducted under very mild conditions (such
as those employed in native chemical ligation; removal of Acm or
other protecting groups that may optionally be used to protect the
thiol groups of other cysteine residues (as with
Hg(CH.sub.2COOH).sub.2); and reverse phase HPLC purification).
Protection of such additional thiol groups prevents oxidative
polymerization of Cys-containing peptides and thus facilitates
their purification and handling.
[0070] B. Extended Native Chemical Ligation
[0071] As indicated above, where the sequence of a natural protein
does not contain a suitable cysteine residue, and it is either
inconvenient or undesirable to modify a natural protein sequence so
as to introduce a cysteine residue at the N-terminus of a
polypeptide, the method of "Extended Native Chemical Ligation" may
be used to ligate polypeptides whose N-terminus has been modified
to contain an N-substituted, and preferably, N.alpha.-substituted,
2 or 3 carbon chain amino alkyl or aryl thiol, and thereby permit
the principles of native chemical ligation to be employed with
polypeptides lacking cysteine residues (see, U.S. Patent
Application Ser. No. 60/231,339, herein incorporated by
reference).
[0072] The method of "Extended Native Chemical Ligation" involves
ligating a first component comprising a carboxyl thioester, and
more preferably, an .alpha.-carboxyl thioester with a second
component comprising an acid stable N-substituted, and preferably,
N.alpha.-substituted, 2 or 3 carbon chain amino alkyl or aryl
thiol. Chemoselective reaction between the carboxythioester of the
first component and the thiol of the N-substituted 2 or 3 carbon
chain alkyl or aryl thiol of the second component proceeds through
a thioester-linked intermediate, and resolves into an initial
ligation product. More specifically, the thiol exchange occurring
between the COSR thioester component and the amino alkyl thiol
component generates a thioester-linked intermediate ligation
product that after spontaneous rearrangement generates an
amide-linked first ligation product through a 5-membered or
6-membered ring intermediate depending upon whether the amino alkyl
thiol component has formula I or II, respectively:
J1-C(O)--N(C1(R1)-C2-SH)-J2 I
J1-C(O)--N(C1(R1)-C2(R2)-C3(R3)-SH)-J2 II where J1 is a peptide or
polypeptide having one or more optionally protected amino acid side
chains, or a moiety of such peptide or polypeptide, a polymer, a
dye, a suitably functionalized surface, a linker or detectable
marker, or any other chemical moiety compatible with chemical
peptide synthesis or extended native chemical ligation; R1, R2 and
R3 are independently H or an electron donating group conjugated to
C1; with the proviso that at least one of R1, R2 and R3 comprises
an electron donating group conjugated to C1; and J2 is a peptide or
polypeptide having one or more optionally protected amino acid side
chains, or a moiety of such peptide or polypeptide, a polymer, a
dye, a suitably functionalized surface, a linker or detectable
marker; or any other chemical moiety compatible with chemical
peptide synthesis or extended native chemical ligation.
[0073] The N-substituted 2 or 3 carbon chain alkyl or aryl thiol
[HS-C2-C1(R1)-] or [HS--(C3(R3)-C2(R2)-C1 (R1)-] at the ligation
site is amenable to being removed, under peptide-compatible
conditions, without damage to the product, to generate a final
ligation product of formula III, having a native amide bond at the
ligation site: J1-C(O)--HN-J2 III where J1, J2, R1, R2, and R3 are
as defined above.
[0074] The R1, R2 and R3 groups are selected to facilitate cleavage
of the N--C1 bond under peptide compatible cleavage conditions. For
example, electron donating groups, particularly if conjugated to
C1, can be used to form a resonance stabilized cation at C1 that
facilitates cleavage. The chemical ligation reaction preferably
includes as an excipient a thiol catalyst, and is carried out
around neutral pH conditions in aqueous or mixed organic-aqueous
conditions. Chemical ligation of the first and second components
may proceed through a five or six member ring that undergoes
spontaneous rearrangement to yield an N-substituted amide linked
ligation product. Where the first and second components are
peptides or polypeptides, the N-substituted amide linked ligation
product has formula IV or V:
J1-C(O)--N.alpha.(C1(R1)-C2-HS)--CH(Z2)-C(O)-J2 IV
J1-C(O)--N.alpha.(C1(R1)-C2(R2)-C3(R3)-HS)-CH(Z2)-C(O)-J2 V where
J1, J2 and R1, R2, R3 are as defined above and Z2 is an amino acid
side chain or a derivative of such side chain.
[0075] The conjugated electron donating groups R1, R2 or R3 of the
N-substituted amide bonded ligation product facilitate cleavage of
the N--Cl bond and removal of the 2 or 3 carbon chain alkyl or aryl
thiol from the N-substituted amide-linked ligation product. Removal
of the alkyl or aryl thiol chain of the N under peptide-compatible
cleavage conditions generates a ligation product having a native
amide bond at the ligation site. Where the first and second
components are peptides or polypeptides, the ligation product will
have the formula: J1-CON.alpha.H--CH(Z2)-C(O)-J2 X
[0076] Exemplary R1 subtituents for Formula I are depicted in Table
H. TABLE-US-00002 TABLE II Formula I ##STR40## R1 Substituent
Groups for Formula I (C1 included for reference) ##STR41##
##STR42##
[0077] Exemplary R1, R2 and R3 substituents for Formula II are
depicted in Table III. TABLE-US-00003 TABLE III Formula II
##STR43## R1, R2 and R3 Substituents (C1 included for reference)
##STR44## ##STR45##
[0078] As with the N-substituted 2 carbon chain compounds,
positioning of the benzyl and picolyl electron-donating
substituents R1', R3' and R5' in the ortho or para positions is
necessary to maintain electronic conjugation to the C1 carbon for
robust cleavage of the N.alpha.-C1 bond following ligation.
However, when R2 and R3 form a benzyl group with C2 and C3, at
least one of R1' and R3' comprises a strong electron donating
group, where R1' or R3' is selected from methoxy (--OCH3), thiol
(--SH), hydroxyl (--OH), and thiomethyl (--SCH3). For the
N-substituted 3 carbon chain thiols in which R2 and R3 are
hydrogens, R1 comprises a benzyl or picolyl group in which R1', R3'
and R5' include either strong or moderate electron-donating groups,
or a combination thereof. As with the N-substituted 2 carbon chain
alkyl or aryl thiols, the strong electron-donating groups enhance
the sensitivity of the 3 carbon chain alkyl or aryl thiol to
cleavage following ligation. Thus a particular electron-donating
group or combination thereof can be selected accordingly.
[0079] Similar to the N-substituted 2 carbon chain compounds, the
N-substituted 3 carbon chain compounds of the present invention may
include a thiol as a substituent of R1 in the R1' and R5' positions
when available for substitution in a construct of interest. Here
again the electron-donating thiol group is conjugated to C1 and its
introduction at these locations enables the compounds to have two
routes for the 6-member ring forming ligation event. It also
increases the local concentration of available thiols for reacting
with the .alpha.-carboxy thioester, and provides for additional
conformations in terms of structural constraints that can improve
ligation.
[0080] Synthesis of the N-terminal N-substituted 2 or 3 carbon
chain alkyl or aryl thiol amino acids of the invention can carried
out as described herein, for example, in Scheme I and Scheme II,
and in accordance with standard organic chemistry techniques known
in the art. See, e.g., "Advanced Organic Chemistry, Reactions,
Mechanisms, and Structure," 4.sup.th Edition, J. March (Ed.), John
Wiley & Sons, New York, N.Y., 1992; "Comprehensive Organic
Transformations, A Guide to Functional Group Preparations," R.
Larock (Ed.), VCH Publishers, New York, N.Y., 1989. They may be
synthesized in solution, by polymer-supported synthesis, or a
combination thereof. The preferred approach employs N alpha
protected N alkylated S-protected amino alkyl- or aryl-thiol amino
acid precursors. The reagents utilized for synthesis can be
obtained from any number of commercial sources. Also, it will be
well understood that the starting components and various
intermediates, such as the individual amino acid derivatives can be
stored for later use, provided in kits and the like.
[0081] In preparing the N-terminal N.alpha.-substituted 2 or 3
carbon chain alkyl or aryl thiol amino acids of the invention,
protecting group strategies are employed. The preferred protecting
groups (PG) utilized in the various synthesis strategies in general
are compatible with Solid Phase Peptide Synthesis ("SPPS"). In some
instances, it also is necessary to utilize orthogonal protecting
groups that are removable under different conditions. Many such
protecting groups are known and suitable for this purpose (See,
e.g., "Protecting Groups in Organic Synthesis", 3rd Edition, T. W.
Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999;
NovaBiochem Catalog 2000; "Synthetic Peptides, A User's Guide," G.
A. Grant, Ed., W. H. Freeman & Company, New York, N.Y.,1992;
"Advanced Chemtech Handbook of Combinatorial & Solid Phase
Organic Chemistry," W. D.. Bennet, J. W. Christensen, L. K.
Hamaker, M. L. Peterson, M. R.Rhodes, and H. H. Saneii, Eds.,
Advanced Chemtech, 1998; "Priciples of Peptide Synthesis, 2nd ed.,"
M. Bodanszky, Ed., Springer-Verlag, 1993; "The Practice of Peptide
Synthesis, 2nd ed.," M. Bodanszky and A. Bodanszky, Eds.,
Springer-Verlag, 1994; and "Protecting Groups," P. J. Kocienski,
Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994). Examples
include benzyloxycarbonyl (Z), Boc, Bpoc, Trt, Nps, FmocCl-Z, Br-Z;
NSC; MSC, Dde, etc. For sulfur moieties, examples of suitable
protecting groups include, but are not limited to, benzyl,
4-methylbenzyl, 4-methoxybenzyl, trityl, ACM, TACAM, xanthyl,
disulfide derivatives, picolyl, and phenacyl.
[0082] More particularly, the Na-substituted 2 or 3 carbon chain
alkyl or aryl thiols can be prepared in accordance with Scheme I
(Solid-Phase preparation of the N.alpha.-substituted precursor),
Scheme II (Solution-Phase preparation of the Na-substituted
precursor ). In Scheme I, N.alpha.-substituted 2 or 3 carbon chain
alkyl or aryl thiols are assembled directly on the solid phase
using standard methods of polymer-supported organic synthesis,
while the N.alpha.-protected, N-alkylated, S-protected, aminoalkyl
or arylthiol amino acid precursor of Scheme II are coupled to the
resin using standard coupling protocols. Where racemic or
diastereomeric products are produced, it may be necessary to
separate these by standard methods before use in extended native
chemical ligation. ##STR46## ##STR47##
[0083] The .alpha.-carboxythioesters can be generated by chemical
or biological methods following standard techniques known in the
art, such as those described herein, including the Examples. For
chemical synthesis, .alpha.-carboxythioester peptides can be
synthesized in solution or from thioester-generating resins, which
techniques are well known (See, e.g., Dawson et al., supra; Canne
et al., supra; Hackeng et al., supra, Aimoto et al.). For instance,
chemically synthesized thioester peptides can be made from the
corresponding peptide .alpha.-thioacids, which in turn, can be
synthesized on a thioester-resin or in solution, although the resin
approach is preferred. The peptide-.alpha.-thioacids can be
converted to the corresponding 3-carboxy-4-nitrophenyl thioesters,
to the corresponding benzyl ester, or to any of a variety of alkyl
thioesters. All of these thioesters provide satisfactory leaving
groups for the ligation reactions, with the 3-carboxy-4-nitrophenyl
thioesters demonstrating a somewhat faster reaction rate than the
corresponding benzyl thioesters, which in turn may be more reactive
than the alkyl thioesters. As another example, a trityl-associated
mercaptoproprionic acid leucine thioester-generating resin can be
utilized for constructing C-terminal thioesters (Hackeng et al.,
supra). C-terminal thioester synthesis also can be accomplished
using a 3-carboxypropanesulfonamide safety-catch linker by
activation with diazomethane or iodoacetonitrile followed by
displacement with a suitable thiol (Ingenito et al., supra;
Bertozzi et al.).
[0084] Ligation of the N-substituted 2 or 3 carbon chain alkyl or
aryl thiol components of the invention with the first
carboxythioester component generates a ligation product having an
N-substituted amide bond at the ligation site. The ligation
conditions of the reaction are chosen to maintain the selective
reactivity of the thioester with the N-substituted 2 or 3 carbon
chain alkyl or aryl thiol moiety. In a preferred embodiment, the
ligation reaction is carried out in a buffer solution having pH
6-8, with the preferred pH range being 6.5-7.5. The buffer solution
may be aqueous, organic or a mixture thereof. The ligation reaction
also may include one or more catalysts and/or one or more reducing
agents, lipids, detergents, other denaturants or solubilizing
reagents and the like. Examples of preferred catalysts are thiol
and phosphine containing moieties, such as thiophenol,
benzylmercaptan, TCEP and alkyl phosphines. Examples of denaturing
and/or solubilizing agents include guanidinium, urea in water or
organic solvents such as TFE, HFIP, DMF, NMP, acetonitrile admixed
with water, or with guanidinium and urea in water. The temperature
also may be utilized to regulate the rate of the ligation reaction,
which is usually between 5.degree. C. and 55.degree. C., with the
preferred temperature being between 15.degree. C. and 40.degree. C.
As an example, the ligation reactions proceed well in a reaction
system having 2% thiophenol in 6M guanidinium at a pH between 6.8
and 7.8.
[0085] For the N-substituted 2 carbon chain alkyl or aryl thiols,
the ligation event results from a thiol exchange that occurs
between the COSR thioester component and the amino alkyl thiol
component. The exchange generates a thioester-linked intermediate
ligation product that after spontaneous rearrangement through a
5-membered ring intermediate generates a first ligation product of
the formula J1-HN--CH(Z1)-C(O)--Na(C1(R1)-C2-SH)--CH(Z2)-J2 having
a removable N-substituted 2 carbon chain alkyl or aryl thiol
[HS--C2-C1(R1)-] at the ligation site, where the substituents are
as defined above. The N-substituted 2 carbon chain alkyl or aryl
thiol [HS--C2-C1(R1)-] at the ligation site is amenable to being
removed, under peptide-compatible conditions, to generate a final
ligation product of the formula J1-HN--CH(Z1)-CO-NH-CH(Z2)-CO-J2
having a native amide bond at the ligation site.
[0086] For the N-substituted 3 carbon chain aryl or alkyl thiols,
the thiol exchange between the COSR thioester component and the
amino alkyl thiol component generates a thioester-linked
intermediate ligation product that after spontaneous rearrangement
through a 6-membered ring intermediate generates a first ligation
product of the formula
J1-HN--CH(Z1)-C(O)--Na(C1-C2(R2)-C3(R3)-SH)--CH(Z2)-J2 having a
removable N-substituted 3 carbon chain alkyl or aryl thiol
[HS--C3(R3)-C2(R2)-C1(R1)-] at the ligation site. The N-substituted
3 carbon chain aryl thiol [HS--C3(R3)-C2(R2)-C1(R1)-] at the
ligation site is amenable to being removed, under
peptide-compatible conditions, to generate a final ligation product
of the formula J1-HN--CH(Z1)-CO--NH--CH(Z2)-CO-J2 having a native
amide bond at the ligation site.
[0087] Removal of the N-substituted alkyl or aryl thiol group is
preferably performed in acidic conditions to facilitate cleavage of
the N--C1 bond, yielding a stabilized, unsubstituted amide bond at
the ligation site. By "peptide-compatible cleavage conditions" is
intended physical-chemical conditions compatible with peptides and
suitable for cleavage of the alkyl or aryl thiol moiety from the
ligation product. Peptide-compatible cleavage conditions in general
are selected depending on the .alpha.-substituted compound
employed, which can be readily deduced through routine and well
known approaches (See, e.g., "Protecting Groups in Organic
Synthesis", 3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John
Wiley & Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic
Peptides, A User's Guide," G. A. Grant, Ed., W.H. Freeman &
Company, New York, N.Y.,1992; "Advanced Chemtech Handbook of
Combinatorial & Solid Phase Organic Chemistry," W. D.. Bennet,
J. W. Christensen, L. K. Hamaker, M. L. Peterson, M. R.Rhodes, and
H. H. Saneii, Eds., Advanced Chemtech, 1998; "Priciples of Peptide
Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-Verlag, 1993; "The
Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky and A.
Bodanszky, Eds., Springer-Verlag, 1994; and "Protecting Groups," P.
J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany,
1994).
[0088] For example, where the R1, R2 or R3 substituents comprises a
methoxy, hydroxyl, thiol or thiomethyl, methyl and the like, the
more universal method for removal involves acidic cleavage
conditions typical for peptide synthesis chemistries. This includes
cleavage of the N--C1 bond under strong acidic conditions or
water-acidic conditions, with or without reducing reagents and/or
scavenger systems (e.g., acid such as anhydrous hydrogen fluoride
(HF), triflouroacetic acid (TFA), or trimethylsulfonyl flouroacetic
acid (TMSFA) and the like). More specific acidic cleavage systems
can be chosen to optimize cleavage of the N.alpha.-C1 bond to
remove the aryl or alkyl thiol moiety for a given construct. Such
conditions are well known and compatible with maintaining the
integrity of peptides. A thiol scavenger may be included,
particularly where tryptophans are present in a peptide or
polypeptide sequence to avoid reaction of the tryptophan side chain
with the liberated aryl or alkyl thiol moiety. Examples of thiol
scavengers include ethanediol, cysteine, beta-mercaptoethanol and
thiocresol.
[0089] Other specialized cleavage conditions include light or
reductive-cleavage conditions when the picolyl group is the
substituent. As an example, when the R1, or R2 and R3 substituents
comprise a picolyl moiety, photolysis (e.g., ultraviolet light),
zinc/acetic acid or electrolytic reduction may be used for cleavage
following standard protocols. Where R1 of the N-substituted 2
carbon chain thiol comprises a thiomethane at R1, the mercury or HF
cleavages can be used. The cleavage system also can be used for
simultaneous cleavage from a solid support and/or as a deprotection
reagent when the first or second ligation components comprise other
protecting groups.
[0090] In one embodiment of the present invention
N.alpha.-substituted 2 or 3 chain alkyl or aryl thiols are employed
in the peptide synthesis step (particularly in automated peptide
synthesis and orthogonal and convergent ligation strategies) to
yield properly N-terminally derivatized polypeptides that can be
used as substrates for extended chemical ligation to yield
synthetic bioactive proteins. Such compounds comprise a fully
protected, partially protected or fully unprotected acid stable
N.alpha.-substituted 2 or 3 carbon chain amino alkyl or aryl thiol
of the formula (PG1)S--C2-C1(R1)-Na(PG2)-CH(Z2)-C(O)-J2 or
(PG1)S--C3(R3)-C2(R2)-C1(R1)-N.alpha.(PG2)-CH(Z2)-C(O)-J2, which
are depicted below in Table IV and Table V. In particular, one or
more of R1, R2 and R3 comprises an electron donating group
conjugated to C1 that, following conversion of the
N.alpha.-substituted amino alkyl or aryl thiol to an
N.alpha.-substituted amide alkyl or aryl thiol, is capable of
forming a resonance stabilized cation at C1 that facilitates
cleavage of the N.alpha.-C1 bond under peptide compatible cleavage
conditions. PG1 and PG2 are protecting groups that are present
individually or in combination or are absent and can be the same or
different, where Z2 is any chemical moiety compatible with chemical
peptide synthesis or extended native chemical ligation, and where
J2 is any chemical moiety compatible with chemical peptide
synthesis or extended native chemical ligation. PG1 (or X1) is a
group for protecting the amine. PG2 (or X2) is a group for
protecting the thiol. Many such protecting groups are known and
suitable for this purpose (See, e.g., "Protecting Groups in Organic
Synthesis", 3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John
Wiley & Sons, Inc., 1999; NovaBiochem Catalog 2000; "Synthetic
Peptides, A User's Guide," G. A. Grant, Ed., W. H. Freeman &
Company, New York, N.Y., 1992; "Advanced Chemtech Handbook of
Combinatorial & Solid Phase Organic Chemistry," W. D.. Bennet,
J. W. Christensen, L. K. Hamaker, M. L. Peterson, M. R.Rhodes, and
H. H. Saneii, Eds., Advanced Chemtech, 1998; "Priciples of Peptide
Synthesis, 2nd ed.," M. Bodanszky, Ed., Springer-Verlag, 1993; "The
Practice of Peptide Synthesis, 2nd ed.," M. Bodanszky and A.
Bodanszky, Eds., Springer-Verlag, 1994; and "Protecting Groups," P.
J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994).
TABLE-US-00004 TABLE IV ##STR48## ##STR49##
[0091] Examples of preferred protecting groups for PG1 and X1
include, but are not limited to [Boc(t-Butylcarbamate),
Troc(2,2,2,-Trichloroethylcarbamate),
Fmoc(9-Fluorenylmethylcarbamate), Br--Z or Cl--Z(Br-- or
Cl-Benzylcarbamate), Dde(4,4,-dimethyl-2,6-dioxocycloex 1-ylidene),
MsZ(4-Methylsulfinylbenzylcarbamate), Msc
(2-Methylsulfoethylcarbamate)
Nsc(4-nitrophenylethylsulfonyl-ethyloxy-carbonyl]. Preferred PG1
and X1 protecting groups are selected from "Protective Groups in
Organic Synthesis," Green and Wuts, Third
Edition,Wiley-Interscience, (1999) with the most preferred being
Fmoc and Nsc. Examples of preferred protecting groups for PG2
include, but are not limited to [Acm(acetamidomethyl), MeoBzl or
Mob(p-Methoxybenzyl), Meb(p-Methylbenzyl),
Trt(Trityl),Xan(Xanthenyl),tButhio(s-t-butyl),Mmt(p-Methoxytrityl),2
or 4 Picolyl(2 or 4
pyridyl)),Fm(9-Fluorenyl-methyl),tbut(t-Butyl),Tacam(Trimethylacetamidome-
thyl)]. Preferred protecting groups PG2 and X2 are selected from
"Protective Groups in Organic Synthesis," Green and Wuts, Third
Edition,Wiley-Interscience, (1999), with the most preferred being
Acm, Mob, MeB, Picolyl TABLE-US-00005 TABLE V ##STR50## ##STR51##
##STR52##
[0092] The protected forms of the N.alpha.-substituted 2 or 3 chain
alkyl or aryl thiols of the invention can be prepared as in Schemes
I and II above.
[0093] The compounds of the present invention may be produced by
any of a variety of means, including halogen-mediated amino
alkylation, reductive amination, and by the preparation of
N.alpha.-protected, N-alkylated, S-protected, amino alkyl- or aryl-
thiol amino acid precursors compatible with solid phase amino acid
synthesis methods. When desirable, resolution of the racemates or
diastereomers produced to give compounds of acceptable chiral
purity can be carried out by standard methods.
II. The Bioactive Peptides and Proteins of the Present
Invention
[0094] A. Synthetic Bioactive Proteins
[0095] The above-described methods can be used to synthesize
synthetic bioactive peptides and proteins. As used herein, a
bioactive protein is said to be "synthetic" if non-recombinant
technology has been employed to polymerize some, and most
preferably all, of its amino acid residues. The term
"non-recombinant technology" is intended to distinguish
technologies that involve organic chemistry and other synthetic
polymerization approaches from technologies involving the
translation of RNA into protein, either in vivo or in vitro. Most
preferably, the synthetic bioactive proteins of the present
invention will be produced in vitro using the principles of organic
chemistry, and in particular the methods of "native chemical
ligation" and "extended native chemical ligation," as discussed
herein. Such chemical synthesis will preferably be conducted using
a convergent synthetic approach in which polypeptide fragments will
be synthesized and then ligated to one another to form the
synthetic bioactive protein. Alternatively, the synthetic bioactive
proteins of the present invention may be synthesized in a single
non-convergent synthesis.
[0096] As indicated above, the synthetic bioactive proteins of the
present invention has substantial molecular weight, preferably
being greater than about 25 kDa. For the purposes of the present
invention, such determinations of molecular weight are to be made
by denaturing SDS polyacrylamide electrophoresis. The term "monomer
molecular weight" is intended to refer to the molecular weight of a
monomer synthetic protein, as distinguished from synthetic proteins
that may possess multiple copies of a protein or polypeptide.
[0097] As used herein, a synthetic protein is said to have
"biological activity" if it possesses a discernible bioactivity
that is dependent upon the synthetic protein's structure or amino
acid sequence, such the variation in such structure or sequence
enhances, modifies, or attenuates the protein's bioactivity.
Without limitation, such "biological activity" includes the
capacity of the protein to mediate a catalytic, signaling or
inducing reaction. As used herein, a protein is said to "mediate a
catalytic reaction" by converting a substrate into a product
without itself being consumed or permanently modified. A protein is
said to "mediate a signaling or inducing reaction" if its presence
causes an organism, or its tissues or cells, to initiate, continue,
enhance, modify, or attenuate gene expression, cellular
differentiation, or cellular proliferation. Examples on such
signaling or inducing reactions include inducing cytokine
expression or a response to cytokine expression, inducing
erythropoiesis, inducing or attenuating inflammation and/or an
inflammatory process, initiating angiogenesis or vascularization,
inducing apoptosis, affecting the cell cycle, etc.
[0098] The bioactivity of the synthetic bioactive proteins of the
present invention also includes the capacity of the protein to bind
specifically to a receptor, ligand or antibody. As used herein, the
term "specific" binding is intended to refer to a
structurally-based binding interaction, such as that existing
between a hormone and its receptor, or an antibody and an antigenic
epitope, as distinguished from "non-specific" binding based upon
charge, solubility, hydrophobicity, etc.
[0099] The synthetic bioactive proteins of the present invention
are preferably synthesized by the condensation of amino acid
residues. Such amino acid residues may be the nucleic acid encoded,
ribosomally installed amino acids: alanine, arginine, asparagine,
aspartate, cysteine, glutamate, glutamine, glycine, histidine,
isoleucine, leucine,, lysine, methionine, phenylalanine, proline,
serine, threonine, tryptophan, tyrosine and valine. In one
embodiment, the amino acid sequence selected for a particular
desired synthetic bioactive protein would be the native, or natural
sequence of that protein. In an alternative embodiment, the
selected amino acid sequence will be composed of, all constructed
from polypeptide fragments that may contain an N-terminal cysteine
residue in place of the amino acid residues present in the native
or natural sequence of that protein. The inclusion of such a
residue permits the polypeptide to be ligated to another
polypeptide (modified to contain a carboxy thioester group) using
the principles of native chemical ligation described here and, and
as such, facilitates the use of convergent synthesis to produce
synthetic bioactive proteins.
[0100] In a further embodiment, the synthetic bioactive proteins of
the present invention may contain "irregular" amino acid residues.
As used herein, the term "irregular amino acid residues is intended
to refer to amino acids that are not encoded by RNA and are not
ribosomally installed. In this regard, the present invention
permits wide selectability and flexibility in the design and/or
construction of synthetic bioactive proteins. Examples of
non-ribosomally installed amino acids that may be used in
accordance with a present invention include: D-amino acids,
.beta.-amino acids, pseudo-glutamate, .gamma.-aminobutyrate,
omithine, homocysteine, N-substituted amino acids (R. Simon et al.,
Proc. Natl. Acad. Sci. U.S.A. (1992) 89: 9367-71; WO 91/19735
(Bartlett et al.), U.S. Pat. No. 5,646,285 (Baindur),
.alpha.-aminomethyleneoxy acetic acids (an amino acid-Gly dipeptide
isostere), and .alpha.-aminooxy acids, etc. Peptide analogs
containing thioamide, vinylogous amide, hydrazino, methyleneoxy,
thiomethylene, phosphonamides, oxyamide, hydroxyethylene, reduced
amide and substituted reduced amide isosteres and
.beta.-sulfonamide(s) may be employed.
[0101] In particular, the use of pseudo-glutamate is advantageous
since the R chain modification permits attachment of polymer
adducts to the synthesized protein. Likewise, N-terminal
N.alpha.-substituted 2 or 3 carbon chain alkyl or aryl thiol amino
acids may be employed. Such residues (where present at the end
terminus or polypeptide) can be advantageously used to ligate that
polypeptide to a polypeptide having a carboxy thioester moiety, in
accordance with the methods of extended native chemical ligation
described herein.
[0102] In one embodiment, all of the amino acid residues of the
synthetic bioactive protein may be joined together by a peptide
bond (i.e., an amide bond). Alternatively, two amino acid residues
(or the C-terminal and N-terminal residues of two polypeptides) may
be linked to one another by a non-amide bond (such as a thioester
bond, an oxime bond, a thioether bond, a directed disulfide bond, a
thiozolidine bond, etc.) (Schnolzer, M. and Kent, S. B. H., Science
(1992) 256:221-225; Rose, K., J. Amer. Chem Soc. (1994) 116:30-33;
Englebretsen, D. R. et al., Tetrahedron Lett. 36:8871-8874; Baca,
M. et al., J. Amer. Chem Soc. (1995) 117:1881-1887; Liu, C. F. et
al., J. Amer. Chem Soc. (1994) 116:4149-4153; Liu, C. F. et al., J.
Amer. Chem Soc. (1996) 118:307-312; Dawson, P. E. et al. (1994)
Science 266:776-779). The invention thus permits a variety of
peptide bond modifications, surrogates and isosteric replacements
to be exploited in the preparation of bioactive proteins.
[0103] The synthetic bioactive proteins of the present invention
may be designed to possess a bioactivity that mimics a bioactivity
associated with a mammalian (including human, simian, bovine,
murine, porcine, ovine, equine, etc.), avian, or piscine protein.
As used herein, a first protein is said to mimic a second protein
if the first protein possesses a bioactivity that is modified,
enhanced, or attenuated with respect to the bioactivity of the
second protein. A bioactivity is said to be "associated" with the
protein if its presence is directly or indirectly dependent upon
the amino acid sequence of the protein.
[0104] In alternative embodiment, the bioactive proteins of the
present invention may be wholly or partly engineered, without
reference to any corresponding natural bioactive counterpart.
Preferred synthetic bioactive proteins of the present invention
include receptors, protein receptor ligands. Examples of such
proteins include adrenocorticotropic hormone receptor and its
bioactive fragments, angiotensin receptor, atrial natriuretic
receptor, bradykininin receptor, growth hormone receptor,
chemotatic receptor, dynorphin receptor, endorphin receptor, the
receptor for .beta.-lipotropin and its bioactive fragments,
enkephalin receptor, enzyme inhibitor receptors, the receptor for
fibronectin and its bioactive fragments, gastrointestinal- and
growth hormone-releasing peptide receptors, the receptor for
luteinizing hormone releasing peptide, the receptor for melanocyte
stimulating hormone, neurotensin receptor, opioid receptor,
oxytocin receptor, vasopressin receptor, vasotocin receptor, the
receptor for parathyroid hormone and fragments, protein kinase
receptor, somatostatin receptor, substance P receptor.
[0105] The synthetic bioactive proteins of the present invention
they also include enzymes, and structural molecules, such as chitin
or collagen, etc.
[0106] In a further embodiment, the synthetic bioactive proteins of
the present invention include cytokines. Cytokines are small
proteins or biological factors (in the range of 5-20 kDa) that are
released by cells and mediate specific effects on cell-cell
interaction, communication and on the behavior of other cells. As
used herein, the term "cytokine" is intended to include the
interleukins ("IL) (such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,
IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, etc.);
lymphokines and signaling molecules such as erythropoiesis
stimulating proteins (e.g., erythropoietin (EPO)), tumor necrosis
factor (TNF), interferons, etc., growth factors, such as
transforming growth factor, nerve growth factor, brain derived
growth factor, neurotrophin-3, neurotrophin-4, heptaocyte growth
factor, T-cell Growth Factor (TGF, TGF-.beta.1, TGF-.beta.2,
TGF-.beta.3, etc.), Colony Stimulating Factors (G-CSF, GM-CSF,
M-CSF etc.), Epidermal Growth Factor (EGF, LIF, KGF, OSM, PDGF,
IGF-1, etc.), Fibroblast Growth Factor (.alpha.FGF, .beta.FGF,
etc.), and hormones, particularly single peptide hormones, such as
growth hormone).
[0107] Cytokines, and particularly chemotactic cytokines, also
comprise particularly preferred examples of proteins whose amino
acid sequence can be used to aid in the design of synthetic
bioactive proteins within the ambit of the present invention.
Cytokines participate in innate and acquired immune responses by
either directly or indirectly mediating leukocyte recruitment, or
by serving as important signals in the activation of leukocytes
that have accumulated at specific sites of microbial invasion. The
pro-inflammatory cytokines, interleukin-1.beta. (IL-1 .beta.) and
interleukin-6, (IL-6) and mediators reflect neutrophil recruitment
and activation, including soluble intercellular adhesion molecule,
interleukin-8 (IL-8) (Kotecha S., European Journal of Pediatrics
(1996) 155 Suppl 2:S14-17). Interleukin-6 is a multifunctional
cytokine primarily involved in the acute-phase response, B cell
differentiation, and antibody production. (Akira S, et al:
Interleukin-6 in biology and medicine. Adv Immunol (1993) 54:1-78).
This cytokine is synthesized and secreted by many different cells,
including monocytes, macrophages, T and B cells, endothelial cells,
and fibroblasts. IL-6 is often induced with the proinflammatory
cytokines TNFalpha and IL-1 in many alarm conditions, and
circulating IL-6 plays an important role in the induction of acute
phase reactions. (Xing Z, et al., J. Clin. Invest. (1998)
101(2):311-20) Cytokines that directly or indirectly influence both
leukocyte recruitment and activation include TNF-alpha,
granulocyte-colony-stimulating factor (G-CSF),
granulocyte-macrophage-colony-stimulating factor (GM-CSF), and
members of the chemokine family. Cytokines that predominantly
regulate the state of leukocyte activation include interferon-gamma
(IFN-gamma), interleukin-10 (IL-10), and interleukin-12 (IL-12).
Cytokines can also influence microbial clearance by inducing or
regulating the expression of other effector molecules, which can
occur in either an autocrine or paracrine fashion.
[0108] Colony-stimulating factors are cytokines produced by a
variety of myeloid and stromal cells that are required for the
proliferation and maturation of hematopoietic stem cells.
Additionally, studies show that these factors augment the effector
cell activities of mature leukocyte populations. Specifically,
G-CSF prolongs neutrophil survival in vitro, augments neutrophil
phagocytic activity, and enhances neutrophil respiratory burst.
(Standiford T J, et al., J. Invest. Med. (19097) 45:335-345). In
contrast, GM-CSF promotes the proliferation and maturation of both
neutrophils and macrophages; the activating properties of this
cytokine are primarily directed toward mature macrophage
populations. (Chen G H, et al, J Immunol (1994) 152:724-734).
[0109] Six closely related subfamilies of chemotactic cytokines,
referred to as chemokines, have now been characterized. Of these,
members of at least two subfamilies contribute to pulmonary
antimicrobial host defense (Mandujano J, et al., Amer. J. Respir.
Crit. Care Med. (1995) 151:1233-1238); Baggiolini M, et al., Ann.
Rev. Immunol. (1997) 15:675-705). Members of the C--X--C chemokine
family, which include IL-8, MIP (macrophage inflammatory
protein)-2, KC, ENA-78, and NAP-2, have predominant neutrophil
stimulatory and chemotactic activities, whereas the C-C family,
which includes MCP (monocyte chemoattractant protein)-1, MCP-2,
MCP-3, RANTES, MIP-1 alpha, and MIP-1 beta, exerts predominant
chemotactic and/or activating effects on macrophages, lymphocytes,
and eosinophils. (Huffnagle G B, et al., The Role Of Chemokines In
Pneumonia, in Koch A, Strieter R, eds. Chemokines in Disease.
Texas: R.G. Landis Co; 1996:151-168)
[0110] Interferon-gamma is a cytokine produced by T cells (both
alpha beta- and gamma delta-T cells) and NK cells that is
instrumental in cell-mediated immunity against a broad spectrum of
pulmonary pathogens. This cytokine activates several macrophage
effector cell functions, including stimulation of respiratory
burst, the ability to present antigen, priming of
macrophage-derived TNF release, and enhanced in vitro macrophage
antimicrobial activity against bacterial, fungal, and mycobacterial
organisms.
[0111] Interleukin-12 is a cytokine that promotes Th1-type immune
responses, while inhibiting Th2-type immune responses.
Specifically, IL-12 stimulates the development of Th1 T cells and
NK cells, and increases the cytolytic activity of CD8+ cells and NK
cells. Most important, IL-12 serves as the major inducer of
IFN-gamma from T cells and NK cells.
[0112] In contrast to IL-12, IL-10 promotes the development of
Th2-type immune responses, while inhibiting cell-mediated
(Th1-type) immunity. (Howard M, et al, J. Clin. Immunol. (1992)
12:239-247). This cytokine was shown to exert potent
anti-inflammatory properties, in part by directly de-activating
neutrophils and macrophages, and by downregulating the expression
of TNF, IFN-gamma, and members of both the C--X--C and C--C
chemokine families. IL-10 is instrumental in attenuating undesired
production of proinflammatory cytokines in states of systemic
immune cell activation.
[0113] EPO is a particularly preferred example of a protein whose
amino acid sequence can be used to aid in the design of a synthetic
bioactive protein having erythropoieis-stimulating activity. EPO is
the principal factor responsible for the regulation of red blood
cell production during steady-state conditions and for accelerating
recovery of red blood cell mass following hemorrhage (Jelkmann, W.
(1992) Physiol. Reviews 72:449; Krantz, S. B. (1991) Blood 77:419;
Porter, D. L. and M. A. Goldberg (1993) Exp. Hematol. 21:399;
Nissenson, A. R. (1994) Blood Purif. 12:6). The circulating form of
human EPO is a 165 amino acid (aa) glycoprotein with a molecular
weight of approximately 30,000 (Sawyer, S. T. et al. (1994)
Hematol. Oncol. Clinics NA 8:895; Jacobs, K. J. et al. Nature
(1985) 313:806; Lin, F-K. et al. Proc. Natl. Acad. Sci. USA (1985)
82:7580). Although the cDNA for EPO predicts a molecule with 166
amino acid residues, the carboxy-terminal arginine is removed in a
post-translational modification (12). There are three potential
sites for N-linked glycosylation and all are filled. One O-linked
carbohydrate moiety is also present (13). The effects of
glycosylation are complex. Although unglycosylated E. coli-derived
EPO shows full biological activity in vitro, glycosylation is
apparently necessary for full activity in vivo. Thus, E.
coli-produced and deglycosylated, naturally derived EPO show very
low activity in animal studies (Krantz, S. B. (1991) Blood 77:419;
Sasaki, H. et al. (1987) J. Biol. Chem. 262:12059; Lowy, P. H. et
al. (1960) Nature 185:102; Wojchowski, D. M. et al. Biochem.
Biophys. Acta 910:224; Dordal, M. S. et al. (1985) Endocrinology
116:2293),
[0114] Consistent with the above, variable glycosylation patterns
show variable effects. For example, desialyated EPO exhibits both
enhanced in vitro and decreased in vivo activity, an effect
attributed to the exposure of galactose residues which are
recognized, bound, and cleared by hepatocytes (Spivak, J. L. and B.
B. Hogans (1989) Blood 73v:90; Goldwasser, E. et al. (1974) J.
Biol. Chem. 249:4202). The branching pattern of fully sialyated EPO
also makes a difference in biological activity. Predominantly
tetra-antennary branched EPO shows activity equivalent to
"standard" EPO, while predominantly bi-antennary branched EPO shows
three-fold more activity in vitro but only 15% of normal activity
in vivo (Takeuchi, M. et al. (1989) Proc. Natl. Acad. Sci. USA
86:7819). Studies have indicated that only N-linked, and not
O-linked sugars, are important in EPO functioning (Higuchi, M. et
al. (1992) J. Biol. Chem. 267:770319).
[0115] Colony-stimulating factors and RANTES also comprise
particularly preferred examples of proteins whose amino acid
sequence can be used to aid in the design of a synthetic bioactive
protein within the ambit of the present invention.
[0116] B. Polymer-Modified Proteins and Polypeptides
[0117] A second attribute of the present invention concerns the
ability to modify a protein or polypeptide to contain one or more
structurally defined polymer adducts at preselected positions.
Although polymer-modified proteins have been previously described,
the nature and manner of the possible polymer-modifications of the
present invention markedly differ from such prior efforts.
[0118] Since the synthetic bioactive proteins of the present
invention are chemically synthesized, in whole or part, it is
possible to synthesize such proteins in a manner that will permit
one to covalently bond polymers (such as polymers or sugar,
polyethylene glycol, glycols, hyaluronic acid, etc.) to one or more
particular, user-selected and user-defined sites in a polypeptide a
protein backbone. Moreover, the synthetic production of the
proteins in the present invention permits one to ensure that such
modifications are present at each of the user-selected and
user-defined sites of every molecule in a preparation. Such
uniformity and control of synthesis markedly distinguishes the
present invention from the random modifications permitted to the
use of the methods of the prior art. Significantly, the present
invention permits one to design a synthetic protein in which any
non-critical residue may be derivatized to contain a polymer
adduct. Moreover, for each such user-selected and user-defined
site, the user may define the precise linkage (amide, thioester,
thioether, oxime, etc.) through which such adducts will be bonded
to the polypeptide or protein backbone. Additionally, the
particular polymer adducts desired to be present at a particular
site may be varied and controlled, such that a final preparation is
obtained in which every protein or polypeptide present contains
precisely the same derivatized adducts at precisely the same
derivatized sites. Thus, the present invention permits the
formation of homogeneous preparations of polymer-modified
polypeptide and proteins.
[0119] In addition to providing a means for varying the position
and number of attachment sites to which polymer can be bound, the
present invention permits one to vary the nature of the bound
polymer. The polymer adducts that may be incorporated into any
particular user-selected and user-defined site can be of any
defined length. Likewise, consistent with the methods of present
invention, it is possible to employ polymers of different lengths
at different sites. Thus, in one embodiment, the synthetic
bioactive proteins of the present invention may be either
mono-modified, or poly-modified, with a polymer adduct. Where more
than one polymer adducts introduced into a particular polypeptide
or protein, the employ polymers may be "mono-speciated,"
"poly-speciated," "uniformly speciated," or to "diversely speciated
in." As used herein, the term "mono-speciated" is intended to refer
to a polypeptide or protein that has been modified by a single
species of polymer. In contrast, the term "poly-speciated" is
intended to refer to a polypeptide or protein that has been
modified by more than a single polymer species. Such poly-speciated
polypeptide or protein are said to be "uniformly-speciated" if, at
each modified site of the polypeptide or protein, the same, single
species of polymer is present. In contrast, a poly-speciated
polypeptide or protein is said to be "diversely-speciated" if, the
modified sites of the polypeptide or protein are modified with
different species of polymer.
[0120] Moreover, it is possible to vary the extent of linearity or
branchedness of the polymer adduct at each of the user-selected in
user-defined sites. Thus the polymer adducts may be linear,
branched, or uniformly branched. The term "uniformly branched" is
intended to mean that all branches of a polymer at a particular
site have the same structure and length. As we appreciate, the
present invention permits one to independently vary both the length
of any individual branch, as well as the structure of the polymer
present at such branchpoint.
[0121] In sum, the present invention permits one to define (1) the
location and (2) frequency of polymer-modified, user-selected and
user-defined sites in a polypeptide or protein backbone, as well as
to control the (3) length, (4) species, and (5) degree of branching
present at each such site. Additionally, with respect to
polypeptide and proteins having multiple polymer modifications, the
present invention permits one to independently define each of the
above-identified five variables for each site. Moreover, with
respect polypeptides and proteins having branched polymer
modifications, the present invention permits one to independently
define each of the above-identified five variables for each
branchpoint. Thus, the present invention provides substantial
flexibility of polymer modification
C. Protein and Peptide Delivery
[0122] The optionally polymer-modified synthetic bioactive
polypeptides and proteins of the present invention may be employed
as pharmaceutical agents to effect the treatment of diseases and
conditions. Most preferably, when administered to a patient or
individual in need of therapy, such synthetic bioactive
polypeptides and/or proteins will be administered using a drug
delivery system. The uses such a system enables a drug to be
presented to the patient in a manner that makes it acceptable for
them and enhances the effectiveness of the desired bioactivity. The
purposes of the present invention, preferred drug delivery systems
include systems capable of administering polypeptides or proteins
buyout oral, nasal, or inhalation routes, or intramuscularly,
subcutaneously, transdermally, intravenously, intraurally or
intraocularly.
[0123] Such drug delivery systems may include formulations that
provide site-specific release, or that enhance protection for the
intestinal mucosa. Suitable formulations include: dry powder
formulations, delivery via viral particle encapsulation, liposome
encapsulation, transdermal patches, electrically aided transport
(electroporation therapy) and polymer/niazone conjugation.
[0124] In a preferred embodiment, such drug delivery devices will
respond to changes in the biological environment and deliver-or
cease to deliverdrugs based on these changes. A range of materials
have been employed to control the release of drugs and other active
agents.: poly(urethanes), poly(siloxanes), poly(methyl
methacrylate), poly(vinyl alcohol) for hydrophilicity and strength,
poly(ethylene), poly(vinyl pyrrolidone), poly(2-hydroxy ethyl
methacrylate), poly(n-vinyl pyrrolidone), poly(methyl
methacrylate), poly(vinyl alcohol), poly(acrylic acid),
polyacrylamide, poly(ethylene-co-vinyl acetate), poly(ethylene
glycol), poly(methacrylic acid), etc. In a further preferred
embodiment, biodegradeable polymers will be employed to facilitate
drug delivery. Such polymers include polylactides (PLA),
polyglycolides (PGA), poly(lactide-co-glycolides) (PLGA),
polyanhydrides, and polyorthoesters. Drug delivery devices, and
methods for their use are described in U.S. Pat. Nos. 6,072,041;
6,041,253; 6,018,678; 6,017,318; 6,002,961; 5,879,712; 5,849,331;
5,792,451; 5,783,212; 5,766,633; 5,759,566; 5,690,954; 5,681,811;
5,654,000; 5,641,511; 5,438,040; 4,810,499; and 4,659,558.
III. The Production of Synthetic Bioactive Proteins
[0125] The production of the synthetic bioactive proteins of the
present invention can be envisioned as having the following steps
or components: design, synthesis, peptide ligation, protein
folding, assessment of bioactivity, polymer modification of the
polypeptide backbone (FIG. 2).
[0126] A. The Design of Preferred Synthetic Bioactive Proteins
[0127] In a particularly preferred embodiment, the synthetic
bioactive proteins of the present invention will be an
erythropoiesis stimulating protein. As used herein, an
erythropoiesis stimulating protein is a protein that mediates the
production of red blood cells. The erythropoiesis stimulating
bioactivity of a protein may be determined by any of a variety of
means, such as by following erythropoiesis after in vivo
administration, by assaying the capacity of the protein to mediate
the proliferation of EPO-dependent cell lines, etc.
[0128] A preferred erythropoiesis stimulating protein is mammalian,
most preferably human, EPO and its analogs. Erythropoietin is
predominantly synthesized and secreted by tubular and juxtatubular
capillary endothelial and interstitial cells of the kidney.
Chronical kidney disease causes the destruction of EPO-producing
cells in the kidney. The resulting lack of EPO frequently induces
anemias. The main clinical use of EPO is therefore the treatment of
patients with severe kidney insufficiency (hematocrit below 0.3)
who usually also receive transfusions as well as the treatment of
anemic patients following chemotherapy. Typically, EPO is
synthesized recombinantly in hamster ovary cells for clinical use.
EPO is a relative heat- and pH-stable acidic (pI=4.5) protein of
165 amino acid residues in its mature form. EPO conveys its
activity through the EPO-receptor. Approximately 40 percent of the
molecular mass of EPO is due to its glycosylation. Glycosylation is
an important factor determining the pharmacokinetic behavior of EPO
in vivo. Non-glycosylated EPO has an extremely short biological
half-life. It still binds to its receptor and may even have a
higher specific activity in vitro. However, recent experiments have
shown that PEGylation of EPO significantly enhances its lifetime,
but to significantly decrease EPO activity in a cell-based assay
system.
[0129] The synthetic erythropoiesis stimulating proteins ("SEPs")
of the present invention are preferably chemically modified
synthetic analogs of human urinary EPO. In a preferred embodiment,
they contain one or more polymer moieties (most preferably pPEG
moieties) attached to one or more peptide residue(s) through a
thioether, oxime, amide or other linkage. In a highly preferred
embodiment, such linkages will be at one or more of the positions
that are naturally glycosylated in human urinary EPO (i.e.,
positions 24, 38, 83 and 126). Most preferably, the SEP molecules
will have pPEG moieties at two or more of such positions. In an
alternative embodiment, other protein residues may be modified by
polymer moieties. The positions of modification for the synthetic
bioactive proteins of the present invention include residues
located in a disordered loop, region or domain of the protein, or
at or near sites of potential protease cleavage. For example,
polymer modifications may be introduced at one or more positions of
9, 69 and/or 125 of EPO. The total molecular weight of the SEP
molecules of the present invention may vary from about 25 to about
150 kDa, and more preferably from about 30 to about 80 kDa. The
molecular weight can be controlled by increasing or decreasing the
number and structure of the polymer (such as PPEG) utilized for
modification of a given analog. The pPEG mediated hydrodynamic MW
for the larger constructs is greater than about 100 kDa. In vitro
assays in cells expressing the human EPO receptor indicate that
SEPs of the present invention have an ED50 that is equivalent to
that of recombinantly produced glycosylated human EPO.
[0130] Oxime-linked pPEG analogs are preferably constructed by
attaching an aminooxy, ketone or aldehyde functionalized pPEG to
the SEP protein at a non-naturally encoded amino acid bearing a
side chain aminooxy, ketone or aldehyde functionality. For example,
positions 89 and 117 of SEP-0 and 1 contain pseudo glutamates (a
non-naturally encoded amino acid bearing a side chain of the
formula --CH.alpha.-CH.sub.2--S--COOH (as compared to glutamate
side chain --CH.alpha.-CH.sub.2--CH.sub.2--COOH)). SEP analogs
utilizing thioether linkages are preferably constructed to contain
a thiol functionality provided by a cysteine or unnatural amino
acid with a side chain bearing the thiol. FIG. 3 depicts the basic
structure of one type of preferred synthetic erythropoiesis
stimulating proteins.
[0131] In an alternative embodiment, the SEP molecules of the
present invention may comprise "circularly permuted" EPO analogs in
which the natural amino and carboxy terminus of EPO have been
relocated. Most preferably, such relocation will move the amino and
carboxy termini to positions of low structural constraint, such as
to disordered loops, etc. The disordered loop around positions 125
and 126 (relative to the native EPO residue numbering system) is an
example of such a relocation site. Most preferably, such SEPs will
be disulfide free, and will be chemically modified to contain
polymer moieties at preselected residues.
[0132] Alternatively, the SEP molecules may have amino and carboxy
termini relocated to a naturally occurring glycosylation site, or
to other sites amenable to glycosylation, such as position 126 and
125. The SEP molecules may also include modifications of the amino
and carboxy termini to eliminate or modify charge (such as by
carboxy amidation, etc.).
[0133] In a preferred example of such circularly permuted
molecules, new N-- and C-termini are provided by positions 126 and
125, respectively. The natural disulfide-forming cysteines at
positions 7, 29, 32 and 161 are preferably replaced by the
non-naturally encoded amino acid, L-.alpha.-N-butyric acid (Aba),
which cannot form disulfide bridges. Residues R166, E37 and V82 are
preferably replaced with alanines to improve production. Also, an
additional cysteine is preferably inserted between positions 1 and
166 relative to the native EPO number scheme, which is numbered
below as `0`. The resulting molecule contains four cysteines (at
positions 126, 0, 38, and 83 (as read in the N-- to C-terminal
direction)), which are utilized as (1) ligation sites and (2)
thioether-forming pPEG attachment sites. Optionally, a cysteine may
replace A125 to provide an additional pPEG attachment site. The
total molecular weight of such SEP molecules of the present
invention may vary from about 25 to about 150 kDa, and more
preferably from about 30 to about 80 kDa. The molecular weight can
be controlled by increasing or decreasing the number and structure
of the polymer (such as pPEG) utilized for modification of a given
analog. The pPEG mediated hydrodynamic MW for the larger constructs
is greater than 100 kDa. Optional pPEG attachment sites are located
at positions 125, 9, and 24 (as read in the N-- to C-terminal
direction). Additional SEP analog designs have alternative N-- and
C-termini in the disordered loop region, and/or truncate residues
from the new N- and/or C-termini. The basic structure of preferred
circularly permuted molecules is shown in FIG. 4.
[0134] In an alternative embodiment, the synthetic bioactive
proteins of the present invention are a mammalian, most preferably
human C-GSF. G-CSF induces the rapid proliferation and release of
neutrophilic granulocytes to the blood stream, and thereby provides
therapeutic effect in fighting infection. The human GCSF protein is
174 amino acids in length (Patent: EP 0459630 (Camble,R. et al.),
and variants of its sequence have been isolated. The protein has
four cysteine residues and an O-glycosylation site at threonine
position 133.
[0135] In one embodiment of such a synthetic GCSF molecules, the
amino acid sequence of the molecule will be altered, relative to
the sequence of native GCSF, so as to contain natural, or more
preferably, non-naturally encoded, hydrophobic residues at one or
more of positions 1, 2 or 3. Optionally, such molecules will be
further modified to contain an additional natural, or more
preferably, non-naturally encoded, hydrophobic residues at position
5 of GCSF, and/or at positions 173 and/or 174.
[0136] In a further preferred embodiment the synthetic GCSF
molecules will be polymer-modified (preferably pPEG) at one or more
of positions 63, 64 and/or 133 and/or at one or more of positions 1
and 174. The pPEG polymers may be linear or branched, charged,
uncharged, or mixed; and may have a molecular weight range of from
about 5 to about 80 kDa, and more preferably, from about 40 to
about 70 kDa pPEG total MW contribution depending on the number and
structure of pPEGs utilized for modification. The hydrodynamic
radius exhibits a larger MW effect in vivo (for example, a 10 kDa
branched pPEG exhibits an effective MW of about 55 kDa). The
estimated pPEG mediated hydrodynamic MW is greater than 100
kDa.
[0137] For analogs utilizing oxime linkage, the pPEG is preferably
attached to non-naturally encoded residues bearing a side chain
aminooxy, ketone or aldehyde functionality. For analogs utilizing
thioether linkage, the pPEG is preferably attached to a cysteine or
unnatural amino acid with a side chain bearing a thiol. For analogs
utilizing amide linkage, the pPEG is attached to a natural or
unnatural amino acid bearing a reactive side chain amine.
[0138] The basic structure of the synthetic bioactive GCSF proteins
is shown in FIG. 5. In the figure, "J" designates a non-naturally
encoded residue having a hydrophobic side chain.
[0139] The bioactivity of such synthetic bioactive GCSF proteins
may be assayed by conventional means, such as by assaying for their
capacity to mediate the proliferation of a factor-dependent human
or mouse cell line (e.g., NFS60 cell line), or by measuring
neutrophil stimulation, half-life, and immunogenicity in vivo,
either with or without a delivery system targeting >20 day
half-life/release).
[0140] In an alternative embodiment, the synthetic bioactive
proteins of the present invention are a mammalian, most preferably
human chemokine, RANTES. RANTES blocks entry of M-tropic HIV
strains through the primary receptor CCR5, and also down-modulates
inflammation pathways involved in asthma, transplant rejection and
wound healing (Kalinkovich A, et al., Immunol Lett. (1999)
68:281-287). The synthetic RANTES molecules of the present
invention preferably differ from the RANTES chemokine in being
chemically modified such that: (1) the N-terminal serine found at
position 1 of RANTES 1-68 is substituted with an n-nonanoyl ("NNY")
group; and (2) the tyrosine at position 3 is substituted with a
non-naturally encoded amino acid having a hydrophobic side chain.
Such compounds have been found to be extremely potent, possessing
ED50 in the .mu.M range, compared to the mM range for recombinant
"Met-RANTES 1-68".
[0141] Potent RANTES analogs have been created having one or more
additional changes to those noted above, such as replacement of the
proline at position 2 with a non-naturally encoded amino acid
having a hydrophobic side chain, and by the attachment of a fatty
acid to the C-terminus of the molecule. Receptor specificity has
been improved in combination with potency by modifications to the
N-loop region corresponding to RANTES residues 12-20. In a further
preferred embodiment, the synthetic RANTES molecules of the
invention incorporate a polymer moiety (such as pPEG, or nonanoy
("NNY") or Y3X at their N-- or C-terminus to improve inter alia in
vivo half-life. The pPEG moiety is preferably attached through an
oxime linkage to a non-naturally encoded amino acid that is
introduced at position 66, 67, 68 or linker at position 68, with
preference to position 67. The fatty acid is preferably attached
through an oxime linkage (preferably through a linker) to residue
68, such as an amino-oxy modified amino acid di- or tri-peptide
linker. Other attachment chemistries may alternatively be employed.
The molecular weight of the larger constructs of such synthetic
RANTES analogs ranges from about 25 kDa to about 45 kDa depending
upon the nature and structure of the pPEG attachment, and for the
larger constructs have a pPEG mediated hydrodynamic MW of greater
than 60 kDa. The structure of preferred synthetic RANTES analogs is
shown in FIG. 6.
[0142] B. Defined and Specific Polymer Modification of the
Synthetic Bioactive Proteins and Peptides
[0143] A further aspect of the present invention relates to
chemical moieties and polymers, in particular water-soluble
polymers, and their use to modify proteins so as to yield protein
drugs with improved properties relative to the unmodified protein.
The anticipated beneficial effects of such modification include but
are not limited to (a) improved potency (b) longer lifetime of the
protein drug in the circulation due to increased proteolytic
stability and reduced clearance rates (c) reduced immunogenicity
and (d) possibly differential targeting compared to the unmodified
molecule.
[0144] As discussed above, although polyethylene glycol has been
employed to modify proteins (see, for example, U.S. Pat. No.
6,027,720, Kuga et al., which relates to the modification of lysine
residues of G-CSF to permit the attachment of polyethylene glycol
molecules via reactive amine groups), such modifications are
associated with molecular heterogeneity, and molecular
diversity.
[0145] As used herein, the term "molecular heterogeneity" is
intended to refer to a variation in the number of polymer molecules
attached to each protein of a protein preparation. The term
"molecular diversity" is intended to refer to (a) a variation in
the site(s) of the protein that are modified by the polymer, (b) a
variation in the length of the polymer adducts of different sites
of the same protein, or of the same site(s) in different proteins
of a protein preparation, or (c) a variation in the extent and/or
nature of any branching of the polymer adducts of different sites
of the same protein, or of the same site(s) in different proteins
of a protein preparation. As used herein, the term "molecularly
homogeneous" is intended to refer to a preparation of a protein in
which all of the protein molecules contain the amino acid sequence
and the same polymer modifications at the same positions. A mixture
of two or more "molecularly homogeneous" protein preparations is
referred to herein as a "molecularly defined" preparation.
[0146] In accordance with this aspect of the invention, a solution
to the above-identified problems of polymer heterogeneity,
diversity, and unsuitability involves the production of a new class
of biocompatible polymers which combine the advantages of both
polypeptides (precise length, convenient synthesis) and "pPEG"
("precision PEG"), a flexible, amphiphilic, non-immunogenic,
polymer not susceptible to proteases) Rose, K. et al. (U.S. patent
application Ser. No. 09/379,297, herein incorporated by reference).
This new class of biocompatible polymer has the formula:
--[CO--X--CO--NH--Y--NH].sub.n-- n is an integer, preferably from
1-100 and more preferably from 2-100, where X and Y are
biocompatible repeat elements of precise structure linked by an
amide bond. Preferably, X and Y will be divalent organic radicals
lacking reactive functional groups or are absent and are the same
or different, and can vary independently with each repeating unit
(n). Preferably, when n=2, at least one of X or Y will be selected
from the group consisting of a substituted, unsubstituted, branched
and linear aliphatic and aromatic group. More preferably, at least
one of X or Y the divalent organic radicals will be selected from
the group consisting of phenyl, a C.sub.1-C.sub.10 alkylene moiety,
a C.sub.1-C.sub.10 alkyl group, a heteroatom-containing phenyl, a
heteroatom-containing C.sub.1-C.sub.10 alkylene moiety, a
heteroatom-containing C.sub.1-C.sub.10 alkyl group, and a
combination thereof.
[0147] Particularly preferred pPEG moieties have the formulae:
--{CO--(CH.sub.2).sub.2--CO--NH--(CH.sub.2).sub.3--(OCH.sub.2CH.sub.2).su-
b.3--CH.sub.2--NH}.sub.n-- where n preferably varies from 1-100 and
more preferably from 2-100; or
--{CO--(CH.sub.2).sub.2--CO--NH--(CH.sub.2).sub.6--NH--CO--(CH.sub.2).sub-
.2--CO--NH--(CH.sub.2).sub.3--(OCH.sub.2CH.sub.2).sub.3--CH.sub.1--NH--}.s-
ub.n--, where n preferably varies from 1-50 and more preferably
from 2-50.
[0148] Such pPEG moieties can be synthesized in any of a variety of
ways. Such moieties are, however, preferably produced using a solid
phase stepwise chain assembly of units, rather than a
polymerization process. The use of such an assembly process permits
the moieties of a preparation to have a defined and homogeneous
structure, as to their length, the nature of their X and Y
substituents, the position(s) (if any) of branch points, and the
length, X and Y substituents, and position(s) of any branches.
[0149] Preferably, such moieties will be synthesized by steps such
as: [0150] (a) acylating the amino or hydroxyl group of a compound
of the formula Z-Q-support with a molar excess of a derivative of a
diacid having the formula, HOOC--X--COOH, where Z is H.sub.2N-- or
HO--; Q is a linker or a target molecule; and the support is a
solid phase, matrix or surface; [0151] (b) activating the free
carboxyl group of the product of step(a); [0152] (c) aminolysing
the product of step (b) with a molar excess of a diamine having the
formula, NH.sub.2--Y--NH.sub.2; and [0153] (d) optionally repeating
steps (a)-(c) using HOOC--X--COOH and NH.sub.2--Y--NH.sub.2, where
said X and Y are divalent organic radicals or are absent and are
the same or different, and can vary independently with each of said
optionally repeated units, and are the same or different from the X
and Y substituents used in any of the previous acylating and
aminolysing steps.
[0154] In preferred embodiments, 6-mers, 12-mers, 18-mers and
32-mers of above repeat unit are employed. Where desired, the
repeat unit can be used, for example, in conjunction with the amino
group of lysine to form branched pPEG structures. The pPEG may be
attached to the synthetic proteins of the present invention by a
variety of chemistries, including thioether, oxime and amide
linkage formation.
[0155] In one embodiment, the solid phase stepwise chain assembly
of units comprises:
[0156] Step 1: Couple protected or unprotected diacid to amino on
resin to generate amide bond at linkage site (where PG is a
protecting group that is present or absent depending on diacid
employed): PG-OOC--X--COOH+NH.sub.2--Y--NH-Resin
[0157] Step 2: Remove protecting group (PG) on resin, if present
HOOC--X--CO--NH--Y--NH-Resin
[0158] Step 3: Couple protected or unprotected diamino to carboxy
on resin to generate amide bond at linkage site (where PG is
present or absent depending on diamino employed)
PG-NH--Y--NH+HOOC--X--CO--NH--Y--NH-Resin
[0159] Step 4: Remove protecting group (PG) on resin, if present
--NH--Y--NH--OC--X--CO--NH--Y--NH-Resin
[0160] Step 5: Repeat steps 1-4 `n` times to add `n` units then
cleave from resin
--[CO--X--CO--NH--Y--NH]--[CO--X--CO--NH--Y--NH]--[CO--X--CO--NH--Y--NH]--
-[CO--X--CO--NH--Y--NH]--
[0161] As discussed, linear and branched pPEG constructs are
preferred water-soluble polymers for attachment to the synthetic
bioactive molecules of the invention. The pPEGs employed bear
pendant groups that are charged or neutral under physiological
conditions, and can be made to vary in attachment chemistry,
length, branching and solubility depending on the pPEG structure
one employs. As noted above, preferred pPEGs of the invention
comprise a water-soluble polyamide having the repeat unit
--CO--X--CO--NH--Y--NH--, where one or both of X and Y comprises a
water-soluble repeat unit, and most preferably a `PEG`-based repeat
unit. Although oligoureas are in principle accessible using a
simple two-step solid phase procedure, as shown below for the case
of an amino resin, NH.sub.2-Resin, and a symmetrical diamine,
NH.sub.2--Y--NH.sub.2: Activation with
carbonyldiimidazole.fwdarw.im-CO--NH-Resin Aminolysis with diamine
NH.sub.2--Y--NH.sub.2.fwdarw.NH.sub.2--Y--NH--CO--NH-Resin where
these two steps may be repeated a number of times to give an
oligourea with repeat unit --NH--Y--NH--CO--, this approach is less
preferred. This is because the yields of the above steps may be
non-quantitative at room temperature, even with very large excesses
of reagents and long reaction times. Accordingly, it is preferable
to use a three-step solid phase procedure, shown below for the case
of an amino resin, NH.sub.2-Resin, to form a polyamide. The
Reagents HO.sub.2C--X--CO.sub.2H and NH.sub.2--Y--NH.sub.2 should
be symmetrical in order to avoid isomeric products. Acylation with
diacid HO.sub.2C--X--CO.sub.2H.fwdarw.HO--CO--X--CO--NH-Resin
Activation with
carbonyldiimidazole.fwdarw.im-CO--OCO--X--CO--NH-Resin Aminolysis
with diamine
NH.sub.2--Y--NH.sub.2.fwdarw.NH.sub.2--Y--NH--CO--X--CO--NH-Resin
[0162] These three steps may be repeated in sequence a number of
times to give a polyamide with repeat unit
--NH--Y--NH--CO--X--CO--. The polymer contains a precise number of
monomer units, X and Y can be varied independently at each step,
and end-groups can be chosen at will. For example, by using
succinic anhydride ("Succ") for the acylation step and
4,7,10-trioxa-1,13-tridecanediamine (also referred to as "EDA" or
"TTD") for the aminolysis step, `PEG`-based polyamides are formed
wherein X is --CH.sub.2CH.sub.2--, Y is
--NH--CH.sub.2CH.sub.2CH.sub.2--(OCH.sub.2CH.sub.2).sub.3--CH.sub.2--NH--
and the repeat unit is
--NH--CH.sub.2CH.sub.2CH.sub.2--(OCH.sub.2CH.sub.2).sub.3--CH.sub.2--NH---
COCH.sub.2CH.sub.2CO--. In spite of the fact that the procedure
involves divalent reagents with no protecting groups, cross-linking
is not a problem when standard commercial peptide synthesis resins
are used (Rose et al., (U.S. patent application Ser. No.
09/379,297); and Rose et al., J. Am. Chem. Soc. (1999) 121:7034),
except as noted below for the case of branched Lys cores for making
branched constructs.
[0163] For example, branched pPEG constructs can be made in a
similar manner as for the "chemobody" constructs described in Rose
et al., (U.S. patent application Ser. No. 09/379,297) and Rose, K.
and Vizzavona, J. (J. Am. Chem. Soc. (1999) 121:7034). Thus,
branched pPEG constructs can be made to have a branching core such
as a lysine branching core, which is coupled through oxime linkers
to a preferred water-soluble polyamide such as
--(COCH.sub.2CH.sub.2CO--NH--CH.sub.2CH.sub.2CH.sub.2--(OCH.sub.2-
CH.sub.2).sub.3--CH.sub.2--NH).sub.n--. For instance, oxime bonds
can readily formed between an aminooxyacetyl group on a Lys side
chain at the other extremity of the polyamide, and glyoxylyl groups
on a lysine core, for example the tetrameric core
(O.dbd.CHCO).sub.4Lys.sub.2Lys-. Thus an oxime linker off of each
lysine branch point can be prepared as the structure
-Lys(COCH.sub.2ON.dbd.CHCO-)amide-. An alternative construction can
place the oxime bond between the polyamide and the free pendant
group of the polyamide, preferably using a monoprotected diamine
and deprotection after coupling the polyamide, to generate a
tetravalent branched constructed depicted below:
[O.dbd.CHCO--(NH--CH.sub.2CH.sub.2CH.sub.2--[OCH.sub.2CH.sub.2].sub.3--CH-
.sub.2--NH--COCH.sub.2CH.sub.2CO-)n].sub.4Lys.sub.2Lys-
[0164] Oxime chemistry can be used in making not only dimeric and
tetrameric branched constructs, but it also suitable for assembling
octameric branched constructs (Rose, K., J. Am. Chem. Soc. (1994)
116:30; Rose, K. et al., Bioconj. Chem. (1996) 7:552). It is, of
course, possible to use other chemistries for such purposes when
using such pPEG polyamides (See, e.g., FIG. 7). Moreover, polyamide
formation may be incorporated into a synthetic scheme for peptide
synthesis involving Boc or Fmoc chemistry, but when elaborating
such schemes it must be borne in mind that the aminolysis step will
remove the Fmoc group, and will remove formyl protection of indole
if Boc chemistry is to be used.
[0165] Accordingly, such pPEGs can be made to have various
branching cores (e.g., Lysine branching core etc.) linked through a
bond of choice (e.g., amide, thioether, oxime etc.) to a linear
water-soluble polyamide (e.g., -Succ-TTD).sub.n-, etc.), where the
free end of each linear water-soluble polyamide can each be capped
with a desired pendant group (e.g., carboxylate, amino, amide etc.)
to provide a desired charge. Moreover the linear and branched pPEGs
of the present invention can be made to comprise a unique
functional group for attachment to a synthetic protein bearing one
or more unique and mutually reactive functional groups, and the
pendant groups of the pPEGs will preferably be non-antigenic (See,
e.g., FIG. 7).
[0166] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference. The discussion of the background to the invention herein
is included to explain the context of the invention. This is not to
be taken as an admission that any of the material referred to was
published, known, or part of the prior art or common general
knowledge anywhere in the world as of the priority date of any of
the claims. Having now generally described the invention, the same
will be more readily understood through reference to the following
examples, which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
EXAMPLE 1
Synthesis of Synthetic Erythropoiesis Stimulating Protein SEP-0
[0167] A synthetic erythropoiesis stimulating protein (SEP) was
synthesized. The sequence of the full-length synthesized protein
(designated "SEP-0 (1-166)" is: TABLE-US-00006 (SEQ ID NO:1)
APPRLICDSR VLERYLLEAK EAEKITTGCA EHCSLNEKIT VPDTKVNFYA WKRMEVGQQA
VEVWQGLALL SEAVLRGQAL LVKSSQPW.psi.P LQLHVDKAVS GLRSLTTLLR
ALGAQK.psi.AIS PPDAASAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR
[0168] where .PSI. denotes a non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group. The SEP-0 protein was synthesized in solution
from four polypeptide segments: TABLE-US-00007 Segment SEP-0:1
(GRFN 1711; composed of residues 1-32 of SEQ ID NO:1): APPRLICDSR
VLERYLLEAK EAEKITTGCA EH-thioester Segment SEP-0:2 (GRFN 1712,
composed of residues 33-88 of SEQ ID NO:1): CSLNEKITVP DTKVNFYAWK
RMEVGQQAVE VWQGLALLSE AVLRGQALLV KSSQPW-thioester (where Cys.sup.33
is Acm protected) Segment SEP-0:3 (GRFN 1713, composed of residues
89-116 of SEQ ID NO:1): CPLQLHVDKA VSGLRSLTTL LRALGAQK-thioester
(where Cys.sup.89 is Acm protected) Segment SEP-0:4 (GRFN 1714,
composed of residues 117-166 of SEQ ID NO:1): CAISPPDAAS AAPLRTITAD
TFRKLFRVYS NFLRGKLKLY TGEACRTGDR-carboxylate (where the C-terminal
cysteine (Cys.sup.161) carries a picolyl (pico) protecting
group)
[0169] The peptides SEP-0:1 and SEP-0:2 and SEP-0:3 were
synthesized on a thioester-generating resin by the in situ
neutralization protocol for Boc (tert-butoxycarbonyl) chemistry and
stepwise solid phase peptide synthesis (SPPS) using established
SPPS, side-chain protection and thioester-resin strategies
(Hackeng, et al., PNAS (1999) 96: 10068-10073; and Schnolzer, et
al., Int. J. Pept. Prot. Res., (1992) 40: 180-193)) on an ABI433A
automated peptide synthesizer or by manual chain assembly, or
ordered and acquired from commercial vendors. For instance, a
standard set of Boc SPPS protecting groups was used, namely:
Arg(Tos); Asp(cHex); Cys(4MeBzl) & Cys(Acm); Glu(cHex);
His(DNP); Lys(ClZ); Ser(Bzl); Thr(Bzl); Trp(formyl); Tyr(BrZ); Met,
Asn, Gln were side-chain unprotected. Segment SEP-0:4 was
synthesized analogously on a --OCH.sub.2-Pam-resin. The peptides
were deprotected and simultaneously cleaved from the resin support
using HF/p-cresol according to standard Boc chemistry procedure;
however, for those peptides containing protecting groups not
removed in HF/p-cresol, the protecting groups were retained. The
peptides were purified by preparative C4 reverse-phase-high
pressure liquid chromatography (HPLC). Fractions containing pure
peptide were identified using ES-MS (electrospray ionization mass
spectrometry), pooled and lyophilized for subsequent ligation.
[0170] Step 1: Ligation #1 Segment SEP-0:4 and segment SEP-0:3 were
dissolved in TFE at 15 mM concentration. Saturated phosphate buffer
(pH 7.9) containing 6 M guanidinium chloride and 1% thiophenol was
added, resulting in a clear solution of the peptide segments. After
ligation, the ligation mix was added to a solution of 2 ml TFE
(trifluoroethanol), 6 ml 6M guanidinium chloride, 100 mM phosphate
containing 25% .beta.-mercaptoethanol and incubated for 20 minutes.
The solution was acidified with a solution of 15 mg/ml TCEP
(tris(2-carboxyethyl)phosphine.HCl) in glacial acetic acid and
loaded onto a preparative C4 reverse-phase HPLC column (1 inch
diameter). The peptides were then purified by preparative gradient
reverse-phase HPLC. Fractions containing the desired ligated
product SEP-0:Acm+SEP-0:3+SEP-0:4 were identified by ES-MS and
pooled.
[0171] Step 2: Acm-removal #1 For Acm removal, the aqueous
acetonitrile solution containing the pooled fractions of
SEP-0:Acm+SEP-0:3+SEP-0:4 was diluted 1.times. with HPLC grade
water, and solid urea was added for a final concentration of 2
molar. A threefold molar excess (relative to the total expected
cysteine concentration) of a 30 mg/ml Hg(acetate).sub.2 solution in
3% aqueous acetic acid was added and the solution is stirred for
one hour. The solution was then made 20% in .beta.-mercaptoethanol,
loaded onto a semi-preparative reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
product SEP-0:3+SEP-0:4 were identified by ES-MS and lyophilized
overnight.
[0172] Step 3: Ligation #2 Equal amounts of SEP-0:3+SEP-0:4 and
SEP-0:2 were jointly dissolved in neat TFE at 15 mM concentration.
250 mM Phosphate buffer (pH 7.5) containing 6 M guanidinium
chloride and 1% thiophenol was added, resulting in a clear solution
of the peptide segments. After one day of ligation, the ligation
mix was added to a solution of 10 ml TFE, 10 ml
.beta.-mercaptoethanol, 10 ml piperidine and 20 ml 6M guanidinium
chloride, pH 4, and incubated for 20 minutes to remove any
remaining protecting groups. The solution was acidified with a
solution of 15 mg/ml TCEP in 20% aqueous acetic acid, loaded onto a
preparative reverse-phase HPLC column and purified with a linear
gradient. Fractions containing the desired ligated product
SEP-O:Acm+SEP-0:2+SEP-0:3+SEP-0:4 were identified by ES-MS and
lyophilized overnight.
[0173] Step 4: Carboxymethylation SEP-0:Acm+SEP-0:2+SEP-0:3+SEP-0:4
was dissolved in TFE at 15 mM concentration. A two-fold excess
(v/v) of 200 mM Phosphate buffer (pH 7.9) containing 6 M
guanidinium chloride was added, resulting in a clear solution of
the peptide segment. A 25-fold excess of bromo-acetic acid
dissolved in a minimum amount of methanol was added, and the
solution was allowed to react for two hours. The solution was
acidified with a solution of 15 mg/ml TCEP in 20% aqueous acetic
acid, loaded onto a preparative reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
carboymethylated product SEP-O:Acm+SEP-0:2+SEP-0:3+SEP-0:4+Et were
identified by ES-MS and pooled.
[0174] Step 5: Picolyl Removal Zinc dust was activated in 2M HCl
for 30 minutes. Peptide-SEP-O:Acm+SEP-0:2+SEP-0:3+SEP-0:4+Et was
dissolved in neat TFE at about 10 mg/ml concentration. The solution
was diluted with 4.times. (v/v relative to TFE) 6M guanidinium
chloride, 100 mM acetate, pH 4, containing (freshly added) 35 mg/ml
L-methionine and 35 mg/ml dodecylsarcosine (i.e. sodium
N-dodecanoylsarcosine). The solution was added to the activated Zn
powder. The reaction was monitored at .about.1 hr intervals and is
complete after five hours. After completion, the supernatant was
removed and the remaining Zn powder washed twice for five minutes
with 6M guanidinium chloride, pH 4, 100 mM acetate containing 35
mg/ml L-methionine and 35 mg/ml dodecylsarcosine containing 20% TFE
as well as once with the same solution containing 20%
.beta.-mercaptoethanol. The combined product was acidified with a
solution of 15 mg/ml TCEP in 20% aqueous acetic acid, loaded onto a
preparative reverse-phase HPLC column and purified with a step
gradient. Fractions containing the desired modified product
SEP-O:Acm+SEP-0:2+SEP-0:3+SEP-0:4+Et-Pico were identified by ES-MS
and pooled.
[0175] Step 6: Acm-removal #2 The pooled solution of
SEP-O:Acm+SEP-0:2+SEP-0:3+SEP-0:4+Et-Pico was diluted 3.times. with
HPLC grade water, and solid urea was added for a final
concentration of 2 molar. A threefold molar excess (relative to the
total expected cysteine concentration) of a 30 mg/ml
Hg(acetate).sub.2 solution in 3% aqueous acetic acid was added and
the solution stirred for one hour. The solution was then made 20%
in .beta.-mercaptoethanol, loaded onto a semi-preparative
reverse-phase HPLC column and purified with a step gradient.
Fractions containing the desired product
SEP-0:2+SEP-0:3+SEP-0:4+Et-Pico were identified by ES-MS, diluted
2.times. (v/v) with water containing 2.times. (w/w relative to
peptide mass) DPC (dodecylphosphocholine) and lyophilized
overnight.
[0176] Step 7: Ligation #3 Equal amounts of
SEP-0:2+SEP-0:3+SEP-0:4+Et-Pico and SEP-0:1 were jointly dissolved
in neat TFE at 15 mM concentration and 250 mM Phosphate buffer (pH
7.5) containing 6 M guanidinium chloride is added. To the solution
1% thiophenol was added. After one day of ligation, the ligation
mix was added to a solution of 10 ml TFE, 10 ml P-mercaptoethanol,
10 ml piperidine and 20 ml 6M guanidinium chloride, pH 4, and
incubated for 20 minutes to remove any remaining protecting groups.
The solution was acidified with a solution of 15 mg/ml TCEP in 20%
aqueous acetic acid, loaded onto a preparative reverse-phase HPLC
column and purified with a linear gradient. Fractions containing
the desired ligated product SEP-0 (1-166): (SEQ ID NO:1) were
identified by electrospray mass spectrometry, diluted 2.times.
(v/v) with water containing 2.times. (w/w relative to peptide mass)
dodecylsarcosine and lyophilized.
[0177] Step 8 Folding: Full-length ligated peptide SEP-0 (1-166)
was dissolved in 200 mM Tris buffer (pH 8.7) containing 6 M
guanidinium chloride and 20% TFE and a ten-fold molar excess
(relative to Cys residues in protein) of cysteine. This solution
was dialyzed overnight against a solution of 200 mM Tris buffer (pH
8.7) containing 3 M guanidinium chloride at room temperature. The
solution was then dialyzed against a solution of 200 mM Tris buffer
(pH 8.7) containing 1 M guanidinium chloride at room temperature
for 4 hours at 4.degree. C. and finally against 10 mM phosphate
buffer (pH 7.0) for 4 hours at 4.degree. C. to yield the final
folded product. Folding was verified by electrospray ES-MS and CD
(circular dichroism) spectrometry.
[0178] Step 9 Purification: The folded polypeptide was concentrated
5.times. in centricon concentrator vials and loaded on to Resource
S cation exchange column equilibrated at 10 mM phosphate, pH 7.0.
The folded protein was eluted in a linear salt gradient to 500 mM
NaCl in 10 minutes. Fractions containing the desired folded product
SEP-0 (1-166) were identified by SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), and frozen and stored at -80.degree. C.
An analytical reverse-phase HPLC chromatogram and an ES-MS spectrum
of the folded protein product as well as a CD spectrum demonstrated
the presence of folded protein.
EXAMPLE 2
Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L30
[0179] A second synthetic erythropoiesis stimulating protein
(designated SEP-1-L30) was synthesized to contain oxime-forming
groups at positions 24 and 126 of SEP-0. These groups were then
used to form SEP-1-L30, in which linear (EDA-Succ-).sub.18
carboxylate (EDA=(4,7,10)-trioxatridecane-1,13diamine, also called
TTD; Succ=--CO--CH.sub.2CH.sub.2CO--) polymers have been joined to
the protein backbone. The sequence of the full-length SEP-1 (1-166)
is: TABLE-US-00008 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK
EAEK.sup.oxITTGCA EHCSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLLALL
SEAVLRGQAL LVKSSQPW.psi.P LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS
PPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR
[0180] where .PSI. denotes an non-native amino acid residue
consisting of a cysteine that is carboxymethylated at the
sulfhydryl group, and where K.sup.ox denotes a non-native lysine
that is chemically modified at the .epsilon.-amino group with an
oxime linker group coupled to a designated water-soluble polymer
through an oxime bond.
[0181] A. Oximation of GRFN1776 and GRFN1711 with GRFNP32
[0182] Oxime formation was performed to attach water-soluble
polymers bearing an aminooxyacetyl group to peptides carrying a
ketone carbonyl group. To accomplish this, the following peptide
segments were synthesized: TABLE-US-00009 Segment SEP-1:4 (GRFN
1776; composed of residues 117-166 of SEQ ID NO:2): CAISPPDAAK
AAPLRTITAD TFRKLFRVYS NFLRGKLKLY TGEACRTGDR-carboxylate (where
Lys.sup.126 is modified with a levulinic acid residue at the
.epsilon.-amino group, and where Cys.sup.117 is Acm protected)
Segment SEP-1:1 (GRFN 1711, composed of residues 1-32 of SEQ ID
NO:2): APPRLICDSR VLERYLLEAK EAEKITTGCA EH-thioester (where Lys24
is modified with a levulinic acid residue)
[0183] Segment SEP-1:1 (GRFN 1711) was synthesized on a
thioester-generating resin, and Segment SEP-1:4 (GRFN 1776) on a
--OCH.sub.2-Pam-resin as in Example 1. Lysines 24 and 126 of these
two peptide segments were initially protected with an Fmoc group at
the .epsilon.-amino group. After completion of the chain assembly,
the Fmoc-bearing amino groups were deprotected following standard
Fmoc deprotection procedures and modified by attachment of
levulinic acid to each peptide resin, respectively. The peptides
were then deprotected and simultaneously cleaved from the resin
support as described in Example 1. The peptides were purified by
preparative C4 reverse phase HPLC. Fractions containing pure
peptide were identified using ES-MS, pooled and lyophilized for
subsequent ligation. GRFNP32 [-(EDA-Succ).sub.18 carboxylate] was
assembled on a Sasrin carboxyl-generating resin following standard
protocols (Rose, K. et al., U.S. patent application Ser. No.
09/379,297; Rose, et al., J Am Chem Soc.(1999) 121: 7034). An
aminooxyacetyl (AoA) moiety was attached to the N-terminal amino
group of the polymer by coupling a fivefold excess of activated
Boc-aminooxyacetic acid. The polymer chain was cleaved from the
resin support using classic Fmoc-chemistry procedures. The polymer
chain was purified by preparative C4 reverse-phase HPLC. Fractions
containing pure polymer were identified using ES-MS, pooled and
lyophilized for subsequent ligation.
[0184] Segment SEP-1:4 and GRFNP32 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unmodified peptide
and unreacted polymer by preparative gradient C4 reverse-phase
HPLC. Fractions containing the desired oximated product
SEP-1:4+GP32 were identified by ES-MS and pooled and
lyophilized.
[0185] Segment SEP-1:1 and GRFNP32 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unreacted polymer
by preparative gradient C4 reverse-phase HPLC. Fractions containing
the desired oximated product SEP-1:1+GP32 were identified by ES-MS
and pooled and lyophilized.
[0186] B. Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L30
[0187] SEP-1-L30 was synthesized in solution from four polypeptide
segments: TABLE-US-00010 Segment SEP-1:1 + GP32 (GRFN 1711 +
GRFNP32, corresponding to residues 1-32 of SEQ ID NO:2): APPRLICDSR
VLERYLLEAK EAEK.sup.oxITTGCA EH-thioester (where Lys.sup.24 is
modified at the .epsilon.-amino group with a levulinic oxime linker
group that is coupled to GRFNP32 through a levulinic-aminooxyacetyl
(Lev- AoA) oxime bond denoted K.sup.ox) Segment SEP-1:2 (GRFN 1712,
corresponding to residues 33-88 of SEQ ID NO:2): CSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW-thioester
(where Cys.sup.33 is Acm protected) Segment SEP-1:3 (GRFN 1713,
corresponding to residues 89-116 of SEQ ID NO:2): CP LQLHVDKAVS
GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89 is Acm protected)
Segment SEP:1:4 + GP32 (GRFN 1776 + GRFNP32, corresponding to
residues 117-166 of SEQ ID NO:2): CAIS PPDAAK.sup.oxAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR-carboxylate (where Lys.sup.126 is
modified at the .epsilon.-amino group with a levulinic oxime linker
group that is coupled to GRFNP32 through a levulinic-aminooxy-
cetyl (Lev-AoA) oxime bond denoted K.sup.ox, and where the
C-terminal cysteine carries a picolyl (pico) protecting group)
[0188] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification were performed as described in Example 1 to yield
full-length, folded SEP-1-L30. An analytical reverse-phase HPLC
chromatogram and an ES-MS spectrum of the folded protein product as
well as a CD spectrum demonstrated the presence of folded
protein
EXAMPLE 3
Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L26
[0189] A third synthetic erythropoiesis stimulating protein
(designated SEP-1-L26) was synthesized to contain oxime-forming
groups at positions 24 and 126 of SEP-0. These groups were then
used to form SEP-1-L26, in which the linear polymers
(EDA-Succ).sub.18 carboxylate and (EDA-Succ).sub.6-amide have been
joined to the protein backbone through oxime linkages at positions
24 and 126, respectively. The sequence of the full-length SEP-1
(1-166) is: TABLE-US-00011 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK
EAEK.sup.oxITTGCA EHCSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL
SEAVLRGQAL LVKSSQPW.psi.P LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS
PPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR
where .PSI. denotes an non-native amino acid residue consisting of
a cysteine that is carboxymethylated at the sulfhydryl group, and
where K.sup.ox denotes a non-native lysine that is chemically
modified at the F-amino group with an oxime linker group coupled to
a designated water-soluble polymer through an oxime bond.
[0190] In contrast to SEP-1-L30, the SEP-1-L26 construct was
designed to bear a smaller and uncharged water-soluble polymer
attached at position 126. The polymer attached at position 24 was
the same as in SEP-1-L30. Assembly of the full-length product was
as described in Example 2.
[0191] A. Oximation of GRFN1776 with GRFNP6 and oximation of
GRFN1711 with GRFNP32
[0192] Oxime formation was performed to attach water-soluble
polymers bearing an aminooxyacetyl group to peptides carrying a
ketone carbonyl group. To accomplish this, the following peptide
segments were synthesized: TABLE-US-00012 Segment SEP-1:4 (GRFN
1776; composed of residues 117-166 of SEQ ID NO:2): CAISPPDAAK
AAPLRTITAD TFRKLFRVYS NFLRGKLKLY TGEACRTGDR-carboxylate (where
Lys.sup.126 is modified with a levulinic acid residue at the
.epsilon.-amino group, and where Cys.sup.117 is Acm protected)
Segment SEP-1:1 (GRFN 1711, composed of residues 1-32 of SEQ ID
NO:2): APPRLICDSR VLERYLLEAK EAEKITTGCA EH-thioester (where
Lys.sup.24 is modified with a levulinic acid residue)
[0193] Segment SEP-1:1 (GRFN 1711) was synthesized on a
thioester-generating resin, and Segment SEP-1:4 (GRFN 1776) on a
--OCH.sub.2-Pam-resin as in Example 1. Lysines 24 and 126 of these
two peptide segments were initially protected with an Fmoc group at
the .epsilon.-amino group. After completion of the chain assembly,
the Fmoc-bearing amino groups were deprotected following standard
Fmoc deprotection procedures and modified by attachment of
levulinic acid to each peptide resin, respectively, following
standard coupling protocols. The peptides were then deprotected and
simultaneously cleaved from the resin support according to standard
Boc-chemistry procedures as in Example 1. The peptides were
separately purified by preparative C4 reverse-phase HPLC. For each
peptide, fractions containing pure peptide were identified using
ES-MS, pooled and lyophilized for subsequent ligation.
[0194] The water soluble polymer (EDA-Succ).sub.18 carboxylate
(GRFNP32) was assembled on a Sasrin carboxy-generating resin
following standard protocols (Rose, K. et al., U.S. patent
application Ser. No. 09/379,297; Rose, et al., J Am Chem Soc.(1999)
121: 7034). The water soluble polymer (EDA-Succ).sub.6-amide
(GRFNP6) was assembled on a Sieber amide-generating resin following
standard protocols (Rose, K. et al., U.S. patent application Ser.
No. 09/379,297; Rose, et al., J Am Chem Soc.(1999) 121: 7034). An
aminooxyacetyl (AoA) moiety was attached to the N-terminal amino
group of each resin-bound polymer by coupling a fivefold excess of
activated Boc-aminooxyacetic acid. The two polymer chains were
separately cleaved from the respective resin supports using classic
Fmoc-chemistry procedures. Each polymer chain was purified by
preparative reverse phase HPLC. For each polymer, fractions
containing pure polymer were identified using ES-MS, pooled and
lyophilized for subsequent ligation.
[0195] Segment SEP-1:4 and GRFNP6 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unmodified peptide
and unreacted polymer by preparative gradient C4 reverse-phase
HPLC. Fractions containing the desired oximated product SEP-1:4+GP6
were identified by ES-MS and pooled and lyophilized.
[0196] Segment SEP-1:1 and GRFNP32 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was then lyophilized. The dried powder was dissolved
and the polymer-modified peptide separated from unreacted polymer
by preparative gradient C4 reverse-phase HPLC. Fractions containing
the desired oximated product SEP-1:1+GP32 were identified by ES-MS
and pooled and lyophilized.
[0197] B. Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-L26
[0198] SEP-1-L26 was synthesized in solution from four polypeptide
segments: TABLE-US-00013 Segment SEP:1:1 + GP32 (GRFN 1711 +
GRFNP32, corresponding to residues 1-32 of SEQ ID NO:2): APPRLICDSR
VLERYLLEAX EAEK.sup.oxITTGCA EH-thioester (where Lys.sup.24 is
modified at the .epsilon.-amino group with a levulinic oxime linker
group that is coupled to GRFNP32 through a levulinic-aminooxyacetyl
(Lev-AoA) oxime bond denoted K.sup.ox) Segment SEP-1:2 (GRFN 1712,
corresponding to residues 33-88 of SEQ ID NO:2): CSLNEKIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LVKSSQPW-thioester
(where Cys.sup.33 is Acm protected) Segment SEP-1:3 (GRFN 1713,
corresponding to residues 89-116 of SEQ ID NO:2): CP LQLHVDKAVS
GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89 is Acm protected)
Segment SEP:1:4 + GP6 (GRFN 1776 + GRFNP6, corresponding to
residues 117-166 of SEQ ID NO:2): CAIS PPDAAK.sup.oxAAPL RTITADTFRK
LFRVYSNFLR GKLKLYTGEA CRTGDR-carboxylate (where Lys.sup.126 is
modified at the .epsilon.-amino group with a levulinic oxime linker
group that is coupled to GRFNP6 through a levulinic-aminooxy-
acetyl (Lev-AoA) oxime bond denoted K.sup.ox, and where the
C-terminal cysteine [i.e. Cys.sup.161] carries a picolyl (pico)
protecting group)
[0199] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification were performed as described in Examples 1 and 2 to
yield full-length, folded SEP-1-L26. An analytical C4 reverse-phase
HPLC chromatogram and an ES-MS spectrum of the folded protein
product as well as a CD spectrum demonstrated the presence of
folded protein.
EXAMPLE 4
Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B50
[0200] A fourth synthetic erythropoiesis stimulating protein
(designated SEP-1-B50) was synthesized. The amino acid sequence of
the full-length SEP-1-B50 is the same as that of SEP-1-L30:
TABLE-US-00014 (SEQ ID NO:2) APPRLICDSR VLERYLLEAK
EAEK.sup.oxITTGCA EHCSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL
SEAVLRGQAL LVKSSQPW.psi.P LQLHVDKAVS GLRSLTTLLR ALGAQK.psi.AIS
PPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR
where .PSI. denotes an non-native amino acid residue consisting of
a cysteine that is carboxymethylated at the sulfhydryl group, and
where K.sup.ox denotes a non-native lysine that is chemically
modified at the .epsilon.-amino group with an oxime linker group
coupled to a designated water-soluble polymer through an oxime
bond.
[0201] However, the protein was derivatized with a branched polymer
construct having four linear (Succ-EDA).sub.12 moieties rather than
the linear (Succ-EDA).sub.18 polymer of SEP-1-L30. Derivatization
was accomplished via oxime linkages. Assembly of the full-length
product was as described in Example 2.
[0202] A. Synthesis of Template GRFNP17 Carrying Multiple Thiol
Groups
[0203] The template GRFNP17 was synthesized manually on an amide
generating (4-methyl)benzhydrylamine(MBHA)-resin on a 0.4 mmol
scale. Fmoc-Lys(Boc)-OH was coupled using standard coupling
protocols (Schnolzer, M., Int J Pept Protein Res. (1992)
40:180-93). 2.1 mmol amino acid, 10% DIEA in 3.8 ml 0.5M HBTU was
used; i.e. 5-fold excess of amino acid. After removal of the Fmoc
protecting group, Fmoc-Lys(Fmoc)-OH was coupled using standard
amino acid coupling protocols (2.1 mmol amino acid, 10% DIEA in 3.8
ml 0.5M HBTU; i.e. 5-fold excess amino acid). After a second Fmoc
removal step, Fmoc-Lys(Fmoc)-OH was coupled using standard amino
acid coupling protocols (4.2 mmol amino acid, 10% DEEA in 7.6 ml
0.5M HBTU; i.e. 5-fold excess amino acid relative to free amine).
After a final Fmoc deprotection step, a five-fold excess (relative
to free amines) of S-acetyl thioglycolic acid pentafluorophenyl
ester (SAMA-oPfp) in DMF was coupled for 30 minutes. The Boc
protecting group of the side chain of the C-terminal lysyl residue
was removed by two times one minute batch washes with neat TFA,
followed by neutralization of the resin by washing with 10% DIEA in
DMF. 2 mmol Boc-aminooxyacetic acid and 2 mmol N-hydroxysuccinimide
(NHS) were dissolved in 3 ml DMF. After addition of 2 mmol DIC
(diisopropylcarbodiimide), the acid was activated for 30-60
minutes. The solution was added to the neutralized resin and
coupled 1 hr. Finally, the S-linked acetyl groups were removed with
20% piperidine in DMF for 30 minutes. The template was deprotected
and simultaneously cleaved from the resin support using HF/p-cresol
according to standard Boc-chemistry procedures in the presence of
cysteine as a scavenger for free aldehyde (Schnolzer, M., Int J
Pept Protein Res. (1992) 40:180-93). The recovered polyamide in 50%
B [i.e. 50% aqueous acetonitrile containing 0.1%TFA] (aldehyde
free) was frozen and lyophilized. For purification, the template
crude product was dissolved in 2 ml 50% B, and 100 ml 100% A [i.e.
0.1% TFA in water] was added to dilute the sample (Avoid
guanidinium chloride or acetate addition, since the addition of
aldehyde is guaranteed). The template was loaded onto a C4
preparative reverse-phase BPLC column equilibrated at T=40.degree.
C. at 3% B. Salts were eluted isocratically and the desired
template, GRFNP17 purified in a linear gradient. Fractions
containing the desired product were identified by ES-MS, pooled and
lyophilized.
[0204] B. Synthesis of Branched Water-Soluble Polymer GRNP29
[0205] GRFNP29, a branched (EDA-Succ).sub.12 polymer of 15 kDa
molecular weight was synthesized by thioether-generating ligation
of purified thiol-containing template GRFNP17 and a linear polymer
GRFNP31, Br-acetyl-(EDA-Succ).sub.12 carboxylate , where GRFNP31
was synthesized on a Sasrin carboxy-generating resin following
standard protocols (Rose, K. et al., U.S. patent application Ser.
No. 09/379,297; Rose, et al., J Am Chem Soc.(1999) 121: 7034).
[0206] A 1.3.times. molar excess (over total thiols) of the
purified GRFNP31, Br-acetylated (EDA-Succ).sub.12, and purified
thiol-containing template GRFNP17 were jointly dissolved 0.1 M
Tris-HCl/6 M guanidinium chloride, pH 8.7 at .about.10 mM
concentration. After dissolution, the solution was diluted
threefold (v/v) with 0.1 M Tris --HCl, pH 8.7 buffer. The ligation
mixture was stirred at room temperature and the reaction monitored
by reversed-phase HPLC and ES/MS. Additional GRFNP31 reactant was
added on an as-needed basis until the desired reaction product was
the major product. For workup, 3.times. (v/v to ligation mix) 0.1 M
acetate/6 M guanidinium chloride, pH 4 was added, and the solution
was loaded onto a preparative C4 reverse-phase HPLC column, and
purified with a linear gradient. Fractions containing pure GRFNP29
construct were identified using ES-MS, pooled and lyophilized.
[0207] C. Oximation of GRFN1776 and GRFN1711 with GRFNP29
[0208] Segments SEP-1:4, and Segment SEP-1:1 were synthesized as
described in Example 2. Segment SEP-1:4 and GRFNP29 were jointly
dissolved at an equimolar ratio in 50% aqueous acetonitrile
containing 0.1% TFA. The solution was lyophilized. The dried powder
was loaded onto a preparative reverse-phase HPLC column (1 inch
diameter). The polymer-modified peptide was separated from
unmodified peptide and unreacted polymer by preparative gradient C4
reverse-phase HPLC. Fractions containing the desired oximated
product SEP:1:4+GP29 were identified by ES-MS and pooled.
[0209] Segment SEP-1:1 and GRFNP29 were jointly dissolved at an
equimolar ratio in 50% aqueous acetonitrile containing 0.1% TFA.
The solution was frozen and lyophilized. The dried powder was
dissolved in 50% aqueous acetonitrile containing 0.1% TFA and
loaded onto a preparative GPC (gel permeation chromatography)
column (1 inch diameter). The polymer-modified peptide was
separated from unmodified peptide and unreacted polymer by
isocratic elution. Fractions containing the desired oximated
product SEP-1:1+GP29 were identified by ES-MS and pooled.
[0210] D. Synthesis Of Synthetic Erythropoiesis Stimulating Protein
SEP-1-B50
[0211] SEP-1-B50 (SEQ ID NO:2) was synthesized in solution from
four polypeptide segments: TABLE-US-00015 Segment SEP-1:1 + GP29
(GRFN 1711 + GRFNP29, corresponding to residues 1-32 of SEQ ID
NO:2): APPRLICDSR VLERYLLEAK EAEK.sup.oxITTGCA EH-thioester (where
Lys.sup.24 is modified at the .epsilon.-amino group with a
levulinic oxime linker group that is coupled to GRFNP29 through a
levulinic-aminooxyacetyl (Lev-AoA) oxime bond denoted K.sup.ox)
Segment SEP-1:2 (GRFN 1712, corresponding to residues 33-88 of SEQ
ID NO:2): CSLNEKIT VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL
LVKSSQPW-thioester (where Cys.sup.33 is Acm protected) Segment
SEP-1:3 (GRFN 1713, corresponding to residues 89-116 of SEQ ID
NO:2): CP LQLHVDKAVS GLRSLTTLLR ALGAQK-thioester (where Cys.sup.89
is Acm protected) Segment SEP11:4 + GP29 (GRFN 1776 + GRFNP29,
corresponding to residues 117-166 of SEQ ID NO:2): CAIS
PPDAAK.sup.oxAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR-carboxylate (where Lys.sup.126 is modified at the
.epsilon.-amino group with a levulinic oxime linker group that is
coupled to GRFNP29 through a levulinic-aminooxy- acetyl (Lev-AoA)
oxime bond denoted K.sup.ox, and where the C-terminal cysteine
carries a picolyl (pico) protecting group)
[0212] Synthesis of additional peptides, ligation reactions,
carboxymethylation, protecting group removal reactions, folding and
purification were performed as described in Examples 1 and 2,
except that purification was on a Resource Q column, to yield
full-length, folded SEP-1-B50 (SEQ ID NO: 2). An analytical C4
reverse-phase HPLC chromatogram and an ES-MS spectrum of the folded
protein product SEP-1-B50 as well as a CD spectrum demonstrated the
presence of folded protein.
EXAMPLE 5
Synthesis of Synthetic Erythropoiesis Stimulating Protein
SEP-3-L42
[0213] A fifth synthetic erythropoiesis stimulating protein
(designated SEP-3-L42) was synthesized. The amino acid sequence of
the full-length SEP-3 protein is: TABLE-US-00016 (SEQ ID NO:3)
APPRLICDSR VLERYLLEAK EAECITTGCA EHCSLNECIT VPDTKVNFYA WKRMEVGQQA
VEVWQGLALL SEAVLRGQAL LACSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS
PPDAACAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA CRTGDR
[0214] The cysteine residues in positions: 24, 38, 83, and 126 were
modified with maleimide-functionalized (EDA-Succ).sub.18 (GRFNP32)
polymer units (via a Michael addition reaction) to form
SEP-3-L42.
[0215] In detail, SEP-3 was synthesized in solution from four
polypeptide segments: TABLE-US-00017 Segment SEP-3:1 (GRFN 1747,
corresponding to residues 1-37 of SEQ ID NO:3): APPRLICDSR
VLERYLLEAK EAECITTGCA EHCSLNE-thioester (where Cys.sup.7,
Cys.sup.29, and Cys.sup.33 are Acm protected) Segment SEP-3:2 (GRFN
1774, corresponding to residues 38-82 of SEQ ID NO:3): CIT
VPDTKVNFYA WKRMEVGQQA VEVWQGLALL SEAVLRGQAL LA-thioester (where
Cys.sup.38 is side chain protected with the Acm group) Segment
SEP-3:3 (GRFN 1749, corresponding to residues 83-125 of SEQ ID
NO:3): CSSQPWEP LQLHVDKAVS GLRSLTTLLR ALGAQKEAIS PPDAA-thioester
(where Cys.sup.83 is side chain protected with the Acm group)
Segment SEP-3:4 (GRFN 1750, corresponding to residues 126-166 of
SEQ ID NO:3): CAAPL RTITADTFRK LFRVYSNFLR GKLKLYTGEA
CRTGDR-carboxylate (where Cys.sup.161 is Pbo [i.e.,
4-(CH.sub.3S(O)-)benzyl-] protected)
[0216] Peptide synthesis. The peptides SEP-3:1 and SEP-3:2 and
SEP-3:3 were synthesized on a thioester-generating resin by the in
situ neutralization protocol for Boc chemistry SPPS, using
established side-chain protection strategies as described in
Example 1, with changes in protecting group strategy as noted in
the specific peptides above. Segment SEP-3:4 was synthesized
analogously on a --OCH.sub.2-Pam-resin. The peptides were
deprotected and simultaneously cleaved from the resin support as
described in Example 1. The resulting peptides described above were
purified by preparative RP-HPLC. Fractions containing pure peptide
were identified using ES-MS, pooled and lyophilized for subsequent
ligation.
[0217] Step 1: Ligation #1. Segment SEP-3:4 and segment SEP-3:3
were dissolved in TFE at 15 mM concentration. Saturated phosphate
buffer (pH 7.5) containing 6 M guanidinium chloride and 1%
thiophenol was added, resulting in a clear solution of the peptide
segments. After ligation, the ligation mix (defined as 1 volume)
was added to 2 volumes of a solution of {2 ml TFE, 6 ml 6 M
guanidinium chloride, 100 mM phosphate containing 25%
.beta.-mercaptoethanol} and incubated for 20 minutes. The solution
was acidified with a solution of 15 mg/ml TCEP in glacial acetic
acid, loaded onto a preparative C4 reverse-phase HPLC column and
purified with a linear gradient. Fractions containing the desired
ligated product SEP-3:Acm+SEP-3:3+SEP-3:4 were identified by ES-MS
and pooled.
[0218] Step 2: Acm-removal #1 For Acm removal, the aqueous
acetonitrile solution containing the pooled fractions of
SEP-3:Acm+SEP-3:3+SEP-3:4 was diluted 1.times. with HPLC grade
water, and solid urea was added for a final concentration of 2
molar. A threefold molar excess (relative to the total expected
cysteine concentration) of a 30 mg/ml Hg(acetate).sub.2 solution in
3% aqueous acetic acid was added and the solution was stirred for
one hour. The solution was then made 20% in .beta.-mercaptoethanol,
loaded onto a semi-preparative C4 reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
ligated product SEP-3:3+SEP-3:4 were identified by ES-MS and
lyophilized overnight.
[0219] Step 3: Ligation #2 Equal amounts of SEP-3:3+SEP-3:4 and
SEP-3.2 were jointly dissolved in neat TFE trifluoroethanol at 15
mM concentration. 250 mM Phosphate buffer (pH 7.5) containing 6 M
guanidinium and 1% thiophenol was added, resulting in a clear
solution of the peptide segments. After one day of ligation, the
ligation mix (defined as 1 volume) was added to 2 volumes of a
solution of 10 ml TFE, 10 ml .beta.-mercaptoethanol, 10 ml
piperidine and 20 ml 6M guanidinium, pH 4, and incubated for 20
minutes to remove any remaining protecting groups. The solution was
acidified with a solution of 15 mg/ml TCEP in 20% aqueous acetic
acid, loaded onto a preparative C4 reverse-phase HPLC column and
purified with a linear gradient. Fractions containing the desired
ligated product SEP-3:Acm+SEP-3:2+SEP-3:3+SEP-3:4 were identified
by ES-MS and lyophilized overnight.
[0220] Step 4 Acm removal For Acm removal, the aqueous acetonitrile
solution containing the pooled fractions of
SEP-3:Acm+SEP-3:2+SEP-3:3+SEP-3:4 was diluted 1.times. with HPLC
grade water, and solid urea added for a final concentration of 2
molar. A threefold molar excess (relative to the total expected
cysteine concentration) of a 30 mg/ml Hg(acetate).sub.2 solution in
3% aqueous acetic acid was added and the solution stirred for one
hour. The solution was then made 20% in .beta.-mercaptoethanol,
loaded onto a C4 semi-preparative reverse-phase HPLC column and
purified with a step gradient. Fractions containing the desired
ligated product SEP-3:2+SEP-3:3+SEP-3:4 were identified by ES-MS
and lyophilized overnight.
[0221] Step 5: Ligation #3 Equal amounts of SEP-3:2+SEP-3:3+SEP-3:4
and SEP:3:1 were jointly dissolved in neat TFE at 15 mM
concentration. 250 mM Phosphate buffer (pH 7.5) containing 6 M
guanidinium and 1% thiophenol was added, resulting in a clear
solution of the peptide segments. After one day of ligation, the
ligation mix (defined as 1 volume) was added to 2 volumes of a
solution of 10 ml TFE, 10 ml .beta.-mercaptoethanol, 10 ml
piperidine and 20 ml 6M guanidinium, pH 4, and incubated for 20
minutes to remove any remaining protecting groups. The solution was
acidified with a solution of 15 mg/ml TCEP in 20% aqueous acetic
acid, loaded onto a preparative C4 reverse-phase HPLC column and
purified with a linear gradient. Fractions containing the desired
ligated product SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4 were identified by
ES-MS and lyophilized overnight.
[0222] Step 6: Attachment of the Polymer GRFNP32. A
maleimide-functionalized linear (EDA-Succ).sub.18 polymer called
GRFNP32-maleimide was prepared by functionalizing GRFNP32 with BMPS
(3-maleimido propionic acid NHS ester, Pierce, USA) following the
manufacturers protocols to form a maleimide-functionalized
(EDA-Succ).sub.18 polymer [i.e., maleimide-(EDA-Succ).sub.18].
SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4 was dissolved in the minimum amount
of TFE required. A threefold excess of GRFNP32-maleimide was
dissolved in 6M guanidinium chloride, 100 mM phosphate, pH 7.5 and
added to the TFE solution. The progress of the Michael addition
reaction was followed by analytical reverse-phase HPLC. After the
reaction was complete, the solution was loaded onto a preparative
C4 reverse-phase HPLC column and purified with a linear gradient.
Fractions containing the desired polymer-modified product
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+pPEG [i.e. the ligated
full-length 166 residue polypeptide chain with four copies of
GRFNP32 attached to the side chain thiols of Cys.sup.24,
Cys.sup.38, Cys.sup.83 and Cys.sup.126 , and thus also called
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+GP32], were identified by
ES-MS and lyophilized overnight.
[0223] Step 7: Pbo removal For Pbo reduction, the lyophilized
powder of SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+GP32 was
dissolved in neat TFA containing 5% ethanedithiol. The Pbo group
was then cleaved by addition of 10% thioanisole and 15%
bromotrimethylsilane for 30 minutes. The solution was dried in a
rotator-evaporator and taken up in aqueous acetonitrile containing
0.1% TFA. The resulting solution was loaded onto a semi-preparative
reverse-phase HPLC column and purified with a step gradient.
Fractions containing the desired Cys.sup.161-deprotected product
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+GP32-Pbo were identified
by ES-MS and lyophilized overnight.
[0224] Step 8: Acm removal For final Acm removal from the side
chains of Cys.sup.7, Cys.sup.29, and Cys.sup.33, the aqueous
acetonitrile solution containing the pooled fractions of
SEP3:Acm+SEP-3:1+SEP-3:2+SEP-3:3+SEP-3:4+GP32-Pbo was diluted
1.times. with HPLC grade water, and solid urea was added for a
final concentration of 2 molar. A threefold molar excess (relative
to the total expected cysteine concentration) of a 30 mg/ml
Hg(acetate).sub.2 solution in 3% aqueous acetic acid was added and
the solution was stirred for one hour. The solution was then made
20% in .beta.-mercaptoethanol, loaded onto a semi-preparative C4
reverse-phase HPLC column and purified with a step gradient.
Fractions containing the desired ligated, polymer-modified product
SEP-3 (1-166) were identified by ES-MS and lyophilized
overnight.
[0225] Step 9 Folding: Full-length ligated, polymer-modified
peptide SEP-3 (1-166) was dissolved in 200 mM Tris buffer (pH 8.7)
containing 6 M guanidinium chloride and 20% TFE and a ten-fold
molar excess (relative to Cys residues in SEP-3) of cysteine. This
solution was dialyzed overnight against a solution of 200 mM Tris
buffer (pH 8.7) containing 3 M guanidinium chloride at room
temperature. The solution was then dialyzed against a solution of
200 mM Tris buffer (pH 8.7) containing 1 M guanidinium chloride for
4 hours at 4.degree. C. and finally against 10 mM phosphate buffer
(pH 7.0) for 4 hours at 4.degree. C. to yield the final folded
product. Folding was verified by electrospray ES-MS and CD
spectrometry.
[0226] Step 10 Purification: The folded polypeptide was
concentrated 5.times. in centricon concentrator vials and loaded on
to Resource S cation exchange column equilibrated at 10 mM
phosphate, pH 7.0. The folded protein was eluted in a linear salt
gradient to 500 mM NaCl in 10 minutes. Fractions containing the
desired folded product SEP-3-L42 were identified by SDS-PAGE, and
frozen and stored at -80.degree. C.
EXAMPLE 6
Bioactivity Assay of Synthetic Erythropoiesis Stimulating
Proteins
[0227] The bioactivity of folded synthetic erythropoiesis
stimulating proteins, SEP-0, SEP-1-L26, SEP-1-L30, SEP-1-B50, and
SEP-3-L42 was determined using UT/7 and 32D 103197 cell lines, in
factor-dependent cell-line proliferation assays using commercial
recombinant erythropoietin as a control standard. UT-7 is a human
megakaryoblastic leukemia cell line with absolute dependence on one
of interleukin-3, granulocyte-macrophage colony-stimulating factor
(GM-CSF), or erythropoietin (EPO) for growth and survival (Miura Y,
et al., Acta Haematol (1998) 99:180-184); Komatsu N, et al., Cancer
Res. (1991) 51:341-8). 32D 103197 is a murine hemopoietic cell line
(Metcalf, D. Int J Cell Cloning (1992) 10:116-25).
[0228] Stock-solutions of the SEP constructs were made in Iscove's
modified Dulbecco's medium (IMDM), 10% FBS (Fetal bovine serum),
glutamine and Penstrep, and serial 2.times. dilutions of these
stock solutions were added to multi-well plates to which human UT/7
EPO cells at a concentration of 5000 cells/50 .mu.l were added. The
plates were incubated at 37.degree. C. in the presence of 5%
CO.sub.2 and monitored daily for growth. After four days, 20 .mu.l
2.5 mg/ml MTT (methylthiazol tetrazolium) in PBS (phosphate
buffered saline) was added and the plates were incubated for four
hours. 150 .mu.l IPA was added and the absorbance of each well was
read at 562 nm. The ED50 (effective dose to reach 50% of maximum
effect) values for the SEP compounds was determined and compared to
that of CHO (Chinese hamster ovary)-cell produced rhEPO
(recombinant human erythropoietin). The results from these
experiments demonstrated that all of the synthetic erythropoiesis
stimulating proteins exhibited bioactivity. ED50 results for SEP-0,
SEP-1-L26, SEP-1-L30, and SEP-1-B50 are shown in Table VI.
TABLE-US-00018 TABLE VI In Vitro ED50 Values (pM) Erythropoiesis
UT-7 32D 103197 Stimulating Protein (Human) Cells (Mouse) Cells
SEP-0 1,570 863 SEP-1-L26 46.5 100.8 SEP-1-L30 71.5 182.5 SEP-1-B50
182 6200 rh EPO 32.5 136.3
[0229] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure as come
within known or customary practice within the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth.
Sequence CWU 1
1
3 1 166 PRT Homo sapiens 1 Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg
Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu Lys
Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu Lys
Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp Lys
Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln Gly
Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70 75 80
Leu Val Lys Ser Ser Gln Pro Trp Cys Pro Leu Gln Leu His Val Asp 85
90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala
Leu 100 105 110 Gly Ala Gln Lys Cys Ala Ile Ser Pro Pro Asp Ala Ala
Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg
Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu
Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg
165 2 166 PRT Homo sapiens 2 Ala Pro Pro Arg Leu Ile Cys Asp Ser
Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu Ala Glu
Lys Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu Asn Glu
Lys Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr Ala Trp
Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55 60 Gln
Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu 65 70
75 80 Leu Val Lys Ser Ser Gln Pro Trp Cys Pro Leu Gln Leu His Val
Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu
Arg Ala Leu 100 105 110 Gly Ala Gln Lys Cys Ala Ile Ser Pro Pro Asp
Ala Ala Lys Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr
Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly
Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly
Asp Arg 165 3 166 PRT Homo sapiens 3 Ala Pro Pro Arg Leu Ile Cys
Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 15 Leu Glu Ala Lys Glu
Ala Glu Cys Ile Thr Thr Gly Cys Ala Glu His 20 25 30 Cys Ser Leu
Asn Glu Cys Ile Thr Val Pro Asp Thr Lys Val Asn Phe 35 40 45 Tyr
Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp 50 55
60 Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu
65 70 75 80 Leu Ala Cys Ser Ser Gln Pro Trp Cys Pro Leu Gln Leu His
Val Asp 85 90 95 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu
Leu Arg Ala Leu 100 105 110 Gly Ala Gln Lys Cys Ala Ile Ser Pro Pro
Asp Ala Ala Cys Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp
Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg
Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr
Gly Asp Arg 165
* * * * *