U.S. patent application number 12/100356 was filed with the patent office on 2008-11-27 for chemically-defined non-polymeric valency platform molecules and conjugates thereof.
This patent application is currently assigned to La Jolla Pharmaceutical Company. Invention is credited to Stephen M. COUTTS, David S. Jones, Douglas Alan Livingston, Lin Yu.
Application Number | 20080293660 12/100356 |
Document ID | / |
Family ID | 26815927 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080293660 |
Kind Code |
A1 |
COUTTS; Stephen M. ; et
al. |
November 27, 2008 |
CHEMICALLY-DEFINED NON-POLYMERIC VALENCY PLATFORM MOLECULES AND
CONJUGATES THEREOF
Abstract
Chemically-defined, non-polymeric valency platform molecules and
conjugates comprising chemically-defined valency platform molecules
and biological or chemical molecules including polynucleotide
duplexes of at least 20 base pairs that have significant binding
activity for human lupus anti-dsDNA autoantibodies.
Inventors: |
COUTTS; Stephen M.; (San
Diego, CA) ; Jones; David S.; (San Diego, CA)
; Livingston; Douglas Alan; (San Diego, CA) ; Yu;
Lin; (San Diego, CA) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Assignee: |
La Jolla Pharmaceutical
Company
San Diego
CA
|
Family ID: |
26815927 |
Appl. No.: |
12/100356 |
Filed: |
April 9, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11542313 |
Oct 2, 2006 |
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12100356 |
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10144391 |
May 10, 2002 |
7115581 |
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11542313 |
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08769041 |
Dec 18, 1996 |
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10144391 |
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08453254 |
May 30, 1995 |
5606047 |
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08769041 |
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08152506 |
Nov 15, 1993 |
5552391 |
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08453254 |
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Current U.S.
Class: |
514/44R ;
530/324; 536/23.1 |
Current CPC
Class: |
A61K 38/00 20130101;
A61K 39/0008 20130101; C07H 21/00 20130101; A61K 47/59 20170801;
A61K 47/645 20170801; A61K 2039/6093 20130101; A61P 17/00 20180101;
Y10S 514/885 20130101; A61K 47/60 20170801; A61K 47/54 20170801;
A61K 39/35 20130101; A61K 47/595 20170801; A61P 29/00 20180101;
C07K 14/003 20130101; A61P 37/00 20180101; A61P 37/06 20180101 |
Class at
Publication: |
514/44 ; 530/324;
536/23.1 |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; C07K 14/435 20060101 C07K014/435; A61P 37/00 20060101
A61P037/00; C07H 21/00 20060101 C07H021/00 |
Claims
1. A conjugate comprising (a) biological or chemical molecules
reacted with (b) a chemically-defined, non-polymeric valency
platform molecule of the formula: G.sup.[1]{T.sup.[1]}.sub.n[1]
Formula 1 or
G.sup.[2]{L.sup.[2]-J.sup.[2]-Z.sup.[2](T.sup.[2]).sub.n[2]}.sub.n[2]
Formula 2 wherein each of G.sup.[1] and G.sup.[2], if present, is
independently a linear, branched or multiply-branched chain
comprising 1-2000 chain atoms selected from the group C, N, O, Si,
P and S; each of the n.sup.[1] moieties shown as T.sup.[1] and each
of the p.sup.[2].times.n.sup.[2] moieties shown as T.sup.[2] is
independently chosen from the group NHR.sup.SUB (amine),
C(.dbd.O)NHNHR.sup.SUB (hydrazide), NHNHR.sup.SUB (hydrazine),
C(.dbd.O)OH (carboxylic acid), C(.dbd.O)OR.sup.ESTER (activated
ester), C(.dbd.O)OC(.dbd.O)R.sup.B (anhydride), C(.dbd.O)X (acid
halide), S(.dbd.O).sub.2X (sulfonyl halide),
C(.dbd.NR.sup.SUB)OR.sup.SUB (imidate ester), NCO (isocyanate), NCS
(isothiocyanate), OC(.dbd.O)X (haloformate),
C(.dbd.O)OC(.dbd.NR.sup.SUB)NHR.sup.SUB (carbodiimide adduct),
C(.dbd.O)H (aldehyde), C(.dbd.O)R.sup.B (ketone), SH (sulfhydryl or
thiol), OH (alcohol), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide), C(.dbd.O)CRB.dbd.CRB2
(.alpha.,.beta.-unsaturated carbonyl), R.sup.ALK--Hg--X (alkyl
mercurial), and S(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2
(.alpha.,.beta.-unsaturated sulfone); wherein each X is
independently a halogen of atomic number greater than 16 and less
than 54 or other good leaving group; each R.sup.ALK is
independently a linear, branched, or cyclic alkyl (1-20C) group;
each R.sup.SUB is independently H, linear, branched, or cyclic
alkyl (1-20C), aryl (6-20C), or alkaryl (7-30C); each R.sup.ESTER
is independently N-hydroxysuccinimidyl, p-nitrophenoxy,
pentafluorophenoxy, or other activating group; each R.sup.B is
independently a radical comprising 1-50 atoms selected from the
group C, H, N, O, Si, P and S; each of the n.sup.[2] moieties shown
as L.sup.[2], if present, is independently chosen from the group O,
NR.sup.SUB and S; each of the n.sup.[2] moieties shown as
J.sup.[2], if present, is independently chosen from the group
C(.dbd.O) and C(.dbd.S); n.sup.[1]=1 to 32; n.sup.[2]=1 to 32;
p.sup.[2]=1 to 8; with the proviso that the product
n.sup.[2].times.p.sup.[2] be greater than 1 and less than 33; each
of the n.sup.[2] moieties shown as Z.sup.[2] is independently a
radical comprising 1-200 atoms selected from the group C, H, N, O,
Si, P and S, containing attachment sites for at least p.sup.[2]
functional groups on alkyl, alkenyl, or aromatic carbon atoms.
2. A conjugate according to claim 1, wherein the biological
molecules comprise polynucleotide duplexes of at least about 20
base pairs each bound to the valency platform molecule, the
duplexes each having a significant binding activity for human
systemic lupus erythematosus anti-dsDNA autoantibodies.
3. A conjugate according to claim 1, wherein the biological or
chemical molecules are selected from the group consisting of
carbohydrates, lipid, lipopolysaccharides, peptides, proteins,
glycoproteins, single-stranded or double-stranded oligonucleotides,
haptens, or chemical analogs thereof such as mimotopes,
aptamers.
4. A conjugate according to claim 1, wherein the biological or
chemical molecules are analogs of immunogens wherein (a) the analog
binds specifically to B cells to which the immunogen binds
specifically and (b) the conjugate lacks a T cell epitope.
5. The conjugate of claim 1, wherein the valency platform molecule
is derivatized by a reagent selected from the group consisting of
DABA, BAHA, BAHA.sub.ox, and AHAB.
6. The conjugate of claim 2, wherein a linker molecule couples the
duplexes to the valency platform molecule.
7. The conjugate of claim 6, wherein the linker molecule is
selected from the group consisting of HAD and HADPS.
8. The conjugate of claim 2, wherein the duplexes are substantially
homogeneous in length.
9. The conjugate of claim 2, wherein the duplexes are substantially
homogeneous in nucleotide composition.
10. The conjugate of claim 2, wherein the duplexes are 20 to 50-bp
in length.
11. The conjugate of claim 2, wherein the duplexes are bound to the
valency platform molecule at or proximate one of their ends.
12. The conjugate of claim 2, wherein the conjugate is a tolerogen
for human systemic lupus erythematosus.
13. A conjugate according to claim 2, wherein the polynucleotide
duplexes have a B-DNA type helical structure and a significant
binding activity for human systemic lupus erythematosus anti-dsDNA
autoantibodies.
14. A pharmaceutical composition for treating lupus comprising the
conjugate of claim 2 formulated with a pharmaceutically acceptable
injectable vehicle.
15. A method for treating an individual for lupus comprising
administering a therapeutically effective amount of the composition
claim 14 to an individual in need of such treatment.
16. A method for making the conjugate of claim 2, comprising: (a)
bonding a multiplicity of single-stranded polynucleotides of at
least about 20 base pairs each on the valency platform molecule;
and (b) annealing complementary single-stranded polynucleotides to
the single-stranded polynucleotides conjugated to the valency
platform molecule to form said duplexes.
17. A pharmaceutical composition for treating an antibody-mediated
pathology comprising a therapeutically effective amount of the
conjugate of claim 2, combined with a pharmaceutically acceptable
carrier.
18. A method of inducing specific B cell anergy to an immunogen in
an individual comprising administering to the individual an
effective amount of the conjugate of claim 17.
19. A method of treating an individual for an antibody-mediated
pathology in which undesired antibodies are produced in response to
an immunogen comprising administering a therapeutically effective
amount of the conjugate of claim 17 to the individual.
20. A method for making a conjugate according to claim 2,
comprising (a) covalently bonding the analog of the immunogen
lacking T cell epitopes to the chemically-defined valency platform
molecule to form a conjugate; and (b) recovering the conjugate from
the reaction mixture.
21. A chemically-defined, non-polymeric valency platform molecule
of the formula:
G.sup.[6]{O--C(.dbd.O)--NR.sup.SUB-Q.sup.[6](T.sup.[6]).sub.p[6-
]}.sub.n[6] Formula 6 or
G.sup.[7]{O--C(.dbd.O)--N[Q.sup.[7](T.sup.[7]).sub.p[7]/2].sub.2}.sub.n[7-
] Formula 7 wherein each of G.sup.[6] and G.sup.[7], if present, is
independently a linear, branched or multiply-branched chain
comprising 1-2000 chain atoms selected from the group C, N, O, Si,
P and S; each of the n.sup.[6].times.p.sup.[6] moieties shown as
T.sup.[6] and each of the n.sup.[7].times.p.sup.[7] moieties shown
as T.sup.[7] is independently chosen from the group NHR.sup.SUB
(amine), C(.dbd.O)NHNHR.sup.SUB (hydrazide), NHNHR.sup.SUB
(hydrazine), C(.dbd.O)OH (carboxylic acid), C(.dbd.O)OR.sup.ESTER
(activated ester), C(.dbd.O)OC(.dbd.O)R.sup.B (anhydride),
C(.dbd.O)X (acid halide), S(.dbd.O).sub.2X (sulfonyl halide),
C(.dbd.NR.sup.SUB)OR.sup.SUB (imidate ester), NCO (isocyanate), NCS
(isothiocyanate), OC(.dbd.O)X (haloformate),
C(.dbd.O)OC(.dbd.NR.sup.SUB)NHR.sup.SUB (carbodiimide adduct),
C(.dbd.O)H (aldehyde), C(.dbd.O)R.sup.B (ketone), SH (sulfhydryl or
thiol), OH (alcohol), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl), R.sup.ALK--Hg--X (alkyl mercurial), and
S(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
sulfone); wherein each X is independently a halogen of atomic
number greater than 16 and less than 54 or other good leaving
group; each R.sup.ALK is independently a linear, branched, or
cyclic alkyl (1-20C) group; each R.sup.SUB is independently H,
linear, branched, or cyclic alkyl (1-20C), aryl (1-20C), or alkaryl
(1-30C); each R.sup.ESTER is independently N-hydroxysuccinimidyl,
p-nitrophenoxy, pentafluorophenoxy, or other activating group; each
R.sup.B is independently a radical comprising 1-50 atoms selected
from the group C, H, N, O, Si, P and S; n.sup.[6]=1 to 32;
P.sup.[6]=1 to 8; with the proviso that the product
n.sup.[6].times.p.sup.[6] be greater than 1 and less than 33;
n.sup.[7]=1 to 32; p.sup.[7]=2, 4, 6 or 8; with the proviso that
the product n.sup.[7].times.p.sup.[7] be greater than 1 and less
than 33; each of the n.sup.[6] moieties shown as Q.sup.[6] and each
of the 2.times.n.sup.[7] moieties shown as Q.sup.[7] is
independently a radical comprising 1-100 atoms selected from the
group C, H, N, O, Si, P and S, containing attachment sites for at
least p.sup.[6] (for Q.sup.[6]) or p.sup.[7]/2 (for Q.sup.[7],
where p.sup.[7]/2 is an integer) functional groups on alkyl,
alkenyl, or aromatic carbon atoms.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/144,391, filed May 10, 2002, which is
continuation of U.S. patent application Ser. No. 08/769,041, filed
Dec. 18, 1996, which is a divisional of U.S. patent application
Ser. No. 08/453,254, filed May 30, 1995, now U.S. Pat. No.
5,606,047, which is a continuation of U.S. patent application Ser.
No. 08/152,506, filed Nov. 15, 1993, now U.S. Pat. No. 5,552,391.
The disclosure of each of these parent applications is incorporated
herein by reference in their entirety.
DESCRIPTION
[0002] 1. Technical Field
[0003] This invention relates to conjugates comprising
chemically-defined, non-polymeric valency platform molecules
coupled to biological or chemical molecules such as polynucleotides
for treating diseases such as the autoimmune disease systemic lupus
erythematosus (SLE or "lupus"). This invention also relates to the
chemically-defined, non-polymeric valency platform molecules.
[0004] 2. Background
[0005] A number of compounds have been employed as carriers for
biologically useful molecules in preparing conjugates that are
alleged to be tolerogenic. For example, Benacerraf, Katz, and their
colleagues investigated and described the use of conjugates of the
random co-polymer D-glutamic acid/D-lysine, referred to as D-GL in
earlier literature (hereinafter D-EK) with haptens and various
antigens to induce specific immune tolerance. See U.S. Pats. Nos.
4,191,668 and 4,220,565.
[0006] Other investigators have studied conjugates of nucleosides
or DNA with other carriers. Borel et al. (Science (1973) 182:76)
evaluated the ability of isogenic mouse IgG-nucleoside conjugates
to reduce the antibody response to denatured DNA in young animals
of the NZB mouse strain. In separate studies Parker et al. (J.
Immunol. (1974) 113:292) evaluated the effect of denatured DNA
conjugated to poly-D-lysine and/or cyclophosphamide on the
progression of the above-described syndrome in NZB mice.
[0007] In a later article (Ann NY Acad Sci (1986) 475:296-306)
Borel et al. describe oligonucleotide-immunoglobulin conjugates.
Borel et al. (J Clin Invest (1988) 82:1901-1907 or U.S. Pat. No.
4,650,675) have described in vitro studies using conjugates of
human immunoglobulin linked to DNA. U.S. Pat. No. 5,126,131
(Dintzis et al.) also relates to conjugates comprising carriers and
molecules involved in immune responses.
[0008] Other references describe conjugates of nonimmunogenic
polymers and immunogens (Sasaki et al., Scand. J. Immun. (1982)
16:191-200; Sehon, Prog. Allergy (1982) 32:161-202; Wilkinson et
al., J. Immunol. (1987) 139:326-331, and Borel et al., J. Immunol.
Methods (1990) 126:159-168).
[0009] In commonly-owned U.S. Ser. Nos. 07/914,869, U.S. Pat. No.
5,162,515, and Ser. No. 07/652,658, conjugates comprising polymeric
carriers such as D-EK, polyethylene glycol, poly-D-lysine,
polyvinyl alcohol, polyvinyl pyrrolidone and immunoglobulins are
described.
[0010] In sum, applicants believe that the prior art shows only
ill-defined chemical compounds or compounds with numerous
non-specific attachment sites employed as valency platform
molecules in conjugates. Because the valency of such compounds, the
specific location of the attachment sites, and the number of
attachment sites are unpredictable and fluctuate widely, prior art
conjugates comprising such compounds cannot be made reproducibly
and show wide ranges in their reported activity.
DISCLOSURE OF THE INVENTION
[0011] In contrast to the above-described art, applicants have
developed conjugates comprising chemically-defined, non-polymeric
valency platform molecules wherein the valency of the platform
molecules is predetermined and wherein each attachment site is
available for binding of a biological or chemical molecule. Valency
platform molecules within the present invention are defined with
respect to their chemical structure, valency, homogeneity and a
defined chemistry which is amenable to effective conjugation with
the appropriate biological and/or synthetic molecules.
[0012] Thus, one aspect of the instant invention is directed to
conjugates comprising the chemically-defined, non-polymeric valency
platform molecules and biological and/or chemical molecules.
Exemplary of biological and/or chemical molecules suitable for
conjugation to chemically-defined, non-polymeric valency platform
molecules to form conjugates within the instant invention are
carbohydrates, drugs, lipids, lipopolysaccharides, peptides,
proteins, glycoproteins, single-stranded or double-stranded
oligonucleotides and chemical analogs thereof, analogs of
immunogens, haptens, mimotopes, aptamers and the like.
Chemically-defined, non-polymeric valency platform molecules
suitable for use within the present invention include, but are not
limited to, derivatives of biologically compatible and
nonimmunogenic carbon-based compounds of the following
formulae:
G.sup.[1]{T.sup.[1]}.sub.n[1] Formula 1
or
G.sup.[2]{L.sup.[2]-J.sup.[2]-Z.sup.[2](T.sup.[2]).sub.n[2]}.sub.n[2]
Formula 2
wherein
[0013] each of G.sup.[1] and G.sup.[2], if present, is
independently a linear, branched or multiply-branched chain
comprising 1-2000, more preferably 1-1000, chain atoms selected
from the group C, N, O, Si, P and S;
[0014] more preferably, G.sup.[2], if present, is a radical derived
from a polyether, a polyamine, or a polyglycol; most preferably,
G.sup.[2] is selected from the group --(CH.sub.2).sub.q-- wherein
q=0 to 20, --CH.sub.2(CH.sub.2OCH.sub.2).sub.rCH.sub.2--, wherein
r=0 to 300, and
C(CH.sub.2OCH.sub.2CH.sub.2--).sub.s(CH.sub.2OH).sub.4-s wherein
s=1 to 4, more preferably s=3 to 4;
[0015] each of the n.sup.[1] moieties shown as T.sup.[1] and each
of the p.sup.[2].times.n.sup.[2] moieties shown as T.sup.[2] is
independently chosen from the group NHR.sup.SUB (amine),
C(.dbd.O)NHNHR.sup.SUB (hydrazide), NHNHR.sup.SUB (hydrazine),
C(.dbd.O)OH (carboxylic acid), C(.dbd.O)OR.sup.ESTER (activated
ester), C(.dbd.O)OC(.dbd.O)R.sup.B (anhydride), C(.dbd.O)X (acid
halide), S(.dbd.O).sub.2X (sulfonyl halide),
C(.dbd.NR.sup.SUB)OR.sup.SUB (imidate ester), NCO (isocyanate), NCS
(isothiocyanate), OC(.dbd.O)X (haloformate),
C(.dbd.O)OC(.dbd.NR.sup.SUB)NHR.sup.SUB (carbodiimide adduct),
C(.dbd.O)H (aldehyde), C(.dbd.O)R.sup.B (ketone), SH (sulfhydryl or
thiol), OH (alcohol), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl), R.sup.ALK--Hg--X (alkyl mercurial), and
S(.dbd.O).sub.2CR.sup.B.dbd.CR.sup.B.sub.2
(.alpha.,.beta.-unsaturated sulfone);
[0016] more preferably each of the n.sup.[1] moieties shown as
T.sup.[1] and each of the p.sup.[2].times.n.sup.[2] moieties shown
as T.sup.[2] is independently chosen from the group NHR.sup.SUB
(amine), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX (alkyl
halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl), R.sup.ALK--Hg--X (alkyl mercurial), and
S(.dbd.O).sub.2CR.sup.B.dbd.CR.sup.B.sub.2
(.alpha.,.beta.-unsaturated sulfone);
[0017] even more preferably each of the n.sup.[1] moieties shown as
T.sup.[1] and each of the p.sup.[2].times.n.sup.[2] moieties shown
as T.sup.[2] is independently chosen from the group NHR.sup.SUB
(amine), C(.dbd.O)CH.sub.2X (haloacetyl), NR.sup.1R.sup.2 wherein
R.sup.1R.sup.2 is --C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide), and
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl);
[0018] most preferably, all of the n.sup.[1] moieties shown as
T.sup.[1] and all of the p.sup.[2].times.n.sup.[2] moieties shown
as T.sup.[2] are identical;
wherein
[0019] each X is independently a halogen of atomic number greater
than 16 and less than 54 or other good leaving group (i.e., weak
bases such as alkyl or alkyl-substituted sulfonates or sulfates and
the like, aryl or aryl-substituted sulfonates or sulfates and the
like that act similarly to a halogen in this setting);
[0020] each R.sup.ALK is independently a linear, branched, or
cyclic alkyl (1-20C) group;
[0021] each R.sup.SUB is independently H, linear, branched, or
cyclic alkyl (1-20C), aryl (6-20C), or alkaryl (7-30C);
[0022] each R.sup.ESTER is independently N-succinimidyl,
p-nitrophenyl, pentafluorophenyl, tetrafluorophenyl,
pentachlorophenyl, 2,4,5-trichlorophenyl, 2,4-dinitrophenyl,
cyanomethyl and the like, or other activating group such as
5-chloro-8-quinolon-1-yl, 1-piperidyl, 1-benzotriazolyl and the
like;
[0023] each R.sup.B is independently a radical comprising 1-50
atoms selected from the group C, H, N, O, Si, P and S;
[0024] each of the n.sup.[2] moieties shown as L.sup.[2], if
present, is independently chosen from the group O, NR.sup.SUB and
S;
[0025] each of the n.sup.[2] moieties shown as J.sup.[2], if
present, is independently chosen from the group C(.dbd.O) and
C(.dbd.S);
[0026] n.sup.[1]=1 to 32, more preferably n.sup.[1]=2 to 16, even
more preferably n.sup.[1]=2 to 8, most preferably n.sup.[1]=2 to
4;
[0027] n.sup.[2]=1 to 32, more preferably n.sup.[2]=1 to 16, even
more preferably n.sup.[2]=1 to 8, yet more preferably n.sup.[2]=1
to 4, most preferably n.sup.[2]=1 to 2;
[0028] p.sup.[2]=1 to 8, more preferably p.sup.[2]=1 to 4, most
preferably p.sup.[2]=1 to 2;
[0029] with the proviso that the product n.sup.[2].times.p.sup.[2]
be greater than 1 and less than 33;
[0030] each of the n.sup.[2] moieties shown as Z.sup.[2] is
independently a radical comprising 1-200 atoms selected from the
group C, H, N, O, Si, P and S, containing attachment sites for at
least p.sup.[2] functional groups on alkyl, alkenyl, or aromatic
carbon atoms;
[0031] more preferably, all of the n.sup.[2] moieties shown as
Z.sup.[2] are identical;
[0032] more preferably, each of the n.sup.[2] moieties shown as
Z.sup.[2] is independently described by a formula chosen from the
group:
Z.sup.[2] is W.sup.[3]--Y.sup.[3] (attachment site).sub.p[2]
Formula 3
Z.sup.[2] is W.sup.[4]--N{Y.sup.[4] (attachment
site).sub.p[2]/2}.sub.2 Formula 4
Z.sup.[2] is W.sup.[5]--CH {Y.sup.[5] (attachment
site).sub.p[2]/2}.sub.2 Formula 5
wherein
[0033] each of the n.sup.[2] moieties shown as W.sup.[3],
W.sup.[4], or W.sup.[5], if present, is independently a radical
comprising 1-100 atoms selected from the group C, H, N, O, Si, P
and S;
[0034] each of the n.sup.[2] moieties shown as Y.sup.[3], each of
the 2.times.n.sup.[2] moieties shown as Y.sup.[4], and each of the
2.times.n.sup.[2] moieties shown as Y.sup.[5] is independently a
radical comprising 1-100 atoms selected from the group C, H, N, O,
Si, P and S, containing attachment sites for at least p.sup.[2]
(for Y.sup.[3]) or p.sup.[2]/2 (for Y.sup.[4] and Y.sup.[5], where
p.sup.[2]/2 is an integer) functional groups on alkyl, alkenyl, or
aromatic carbon atoms;
[0035] more preferably, each of the n.sup.[2] moieties shown as
W.sup.[3], if present, is independently chosen from the group
(CH.sub.2).sub.r, (CH.sub.2CH.sub.2O).sub.r, NR.sup.SUB
(CH.sub.2CH.sub.2O).sub.rCH.sub.2CH.sub.2, and
NR.sup.SUB(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O), wherein r=1 to
10;
[0036] more preferably, each of the n.sup.[2] moieties shown as
Y.sup.[3] is independently linear, branched, or cyclic alkyl
(1-20C), aryl (6-20C), or alkaryl (7-30C); most preferably, each of
the n.sup.[2] moieties shown as Y.sup.[3] is independently chosen
from the group C.sub.6H.sub.4 (phenyl-1,4-diradical),
C.sub.6H.sub.3 (phenyl-1,3,5-triradical), and (CH.sub.2).sub.r
wherein r=1 to 10;
[0037] more preferably, each of the n.sup.[2] moieties shown as
W.sup.[4], if present, is independently chosen from the group
(CH.sub.2).sub.rC(.dbd.O) and (CH.sub.2).sub.rNR.sup.SUBC(.dbd.O),
wherein r=1 to 10;
[0038] more preferably, each of the 2.times.n.sup.[2] moieties
shown as Y.sup.[4], is independently chosen from the group
(CH.sub.2).sub.r, (CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)
(CH.sub.2).sub.q,
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)
(CH.sub.2).sub.qNR.sup.SUBC(.dbd.O) (CH.sub.2).sub.r,
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.qNR.sup.SUBC(.dbd.O)
(CH.sub.2).sub.r, (CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)
(CH.sub.2CH.sub.2O).sub.qCH.sub.2CH.sub.2, and
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB
(CH.sub.2CH.sub.2O).sub.qCH.sub.2CH.sub.2, wherein r=1 to 10, more
preferably r=2 to 6, and q=1 to 10, more preferably q=1 to 3;
[0039] more preferably, each of the n.sup.[2] moieties shown as
W.sup.[5], if present, is independently chosen from the group
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB and
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)NR.sup.SUB, wherein r=1 to
10;
[0040] more preferably, each of the 2.times.n.sup.[2] moieties
shown as Y.sup.[5], is independently chosen from the group
(CH.sub.2).sub.r and
(CH.sub.2).sub.nC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q, wherein r=1 to
10 and q=1 to 10.
[0041] In a further preferred embodiment for treating lupus, a
conjugate comprises a chemically-defined, non-polymeric valency
platform molecule and a multiplicity of polynucleotide duplexes of
at least about 20 base pairs each bound to the platform molecule,
and having significant binding activity for human SLE anti-dsDNA
autoantibodies. In these preferred embodiments, the polynucleotide
duplexes are substantially homogeneous in length and one strand of
the duplex is conjugated to the valency platform molecule either
directly or via a linker molecule. Usually synthetic
polynucleotides are coupled to a linker molecule before being
coupled to a valency platform molecule. Usually the linker
containing strand of the duplex is coupled at or proximate (i.e.
within about 5 base pairs) one of its ends such that each strand
forms a pendant chain of at least about 20 base pairs measured from
the site of attachment of the strand to the linker molecule. The
second strand is then annealed to the first strand to form a
duplex. Thus, a conjugate within the present invention can be
generally described by the following formula:
[(PN).sub.n-linker].sub.m-valency platform molecule.
wherein PN=a double stranded polynucleotide with "n" nucleotides,
wherein n=at least about 20, and m=2-8.
[0042] Exemplary of suitable linker molecules within the present
invention are 6 carbon thiols such as HAD, a thio-6 carbon chain
phosphate, and HAD.sub.pS, a thio-6 carbon chain phosphorothioate.
Chemically-defined valency platform molecules within the present
invention are formed, for example, by reacting amino modified-PEG
with 3,5-bis-(iodoacetamido) benzoyl chloride (hereinafter "IA
DABA");
3-carboxypropionamide-N,N-bis-[(6'-N'-carbobenzyloxyaminohexyl)acetamide]-
4''-nitrophenyl ester (hereinafter "BAHA");
3-carboxypropionamide-N,N-bis-[(8'-N'-carbobenzyloxyamino-3',6'-dioxaocty-
l)acetamide]4''-nitrophenyl ester (hereinafter "BAHA.sub.ox"); or
by reacting PEG-bis-chloroformate with
N,N-di(2-[6'-N'-carbobenzyloxyaminohexanoamido]ethyl)amine
(hereinafter "AHAB") to form chemically-defined valency platform
molecules.
[0043] Surprisingly unexpected results of at least approximately
ten fold up to more than one-hundred fold increase in
immunosuppression are achieved using conjugates comprising the
chemically-defined, non-polymeric valency platform molecules of the
instant invention and biological or synthetic molecules
(non-haptens) when compared to the polymeric carriers described in
the prior art. For example, at least a one hundred-fold increase in
the immunosuppression of anti-dsDNA autoantibodies was achieved as
described herein using conjugates within the present invention
comprising chemically-defined, non-polymeric valency platform
molecules when compared to conjugates comprising an ill-defined
carrier described in the prior art.
[0044] Still another aspect is a conjugate of (a) a
chemically-defined, non-polymeric valency platform molecule and (b)
a multiplicity of polynucleotide duplexes each and all of which is
bound to the valency platform molecule by a functional group
located at or proximate a terminus of one of the strands of the
duplex, said conjugate being a human SLE tolerogen.
[0045] Pharmaceutical compositions of the above-described
conjugates and pharmaceutically acceptable vehicles are another
aspect of the invention.
[0046] A further aspect of the invention is a method for treating
SLE in an individual in need of such treatment comprising
administering to the individual an effective amount of the
above-described conjugates.
[0047] Yet another aspect of the invention is a method of inducing
specific B cell anergy to an immunogen in an individual comprising
administering to the individual an effective amount of the
above-described conjugates.
[0048] Another aspect of the invention is a method of treating an
individual for an antibody-mediated pathology in which undesired
antibodies are produced in response to an immunogen comprising
administering to the individual an effective amount of the
above-described conjugates.
[0049] A further aspect of the invention is a method for making the
conjugates described above comprising: covalently bonding the
biological or chemical molecule to a chemically-defined valency
platform molecule to form a conjugate.
[0050] A further aspect of the invention is a method for making the
conjugates for treating SLE described above comprising: reacting a
multiplicity of single-stranded polynucleotides each of which is at
least about 20 nucleotides in length and has a functional group at
or proximate one of its termini that reacts with functional groups
on the chemically-defined valency platform molecule to form a
conjugate, and annealing complementary single-stranded
polynucleotides to the single-stranded polynucleotides conjugated
to the chemically-defined valency platform molecule to form pendant
chains of double-stranded DNA.
[0051] Yet another aspect of the invention is directed to novel
chemically-defined, non-polymeric valency platform molecules of the
formulae:
G.sup.[6]{O--C(.dbd.O)--NR.sup.SUB-Q.sup.[6](T.sup.[6]).sub.p[6]}.sub.n[-
6] Formula 6
or
G.sup.[7]{O--C(.dbd.O)--N[Q.sup.[7](T.sup.[7]).sub.p[7]/2].sub.2}.sub.n[-
7] Formula 7
wherein
[0052] each of G.sup.[6] and G.sup.[7], if present, is
independently a linear, branched or multiply-branched chain
comprising 1-2000, more preferably 1-1000, chain atoms selected
from the group C, N, O, Si, P and S; more preferably, each of
G.sup.[6] and G.sup.[7] is a radical derived from a polyalcohol, a
polyamine, or a polyglycol; most preferably, each of G.sup.[6] and
G.sup.[7] is selected from the group --(CH.sub.2).sub.q-- wherein
q=0 to 20, --CH.sub.2(CH.sub.2OCH.sub.2).sub.rCH.sub.2--, wherein
r=0 to 300, and
C(CH.sub.2OCH.sub.2CH.sub.2--).sub.s(CH.sub.2OH).sub.4-s, wherein
s=1 to 4, more preferably s=3 to 4;
[0053] each of the n.sup.[6].times.p.sup.[6] moieties shown as
T.sup.[6] and each of the n.sup.[7].times.p.sup.[7] moieties shown
as T.sup.[7] is independently chosen from the group NHR.sup.SUB
(amine), C(.dbd.O)NHNHR.sup.SUB (hydrazide), NHNHR.sup.SUB
(hydrazine), C(.dbd.O)OH (carboxylic acid), C(.dbd.O)OR.sup.ESTER
(activated ester), C(.dbd.O)OC(.dbd.O)R.sup.B (anhydride),
C(.dbd.O)X (acid halide), S(.dbd.O).sub.2X (sulfonyl halide),
C(.dbd.NR.sup.SUB)OR.sup.SUB (imidate ester), NCO (isocyanate), NCS
(isothiocyanate), OC(.dbd.O)X (haloformate),
C(.dbd.O)OC(.dbd.NR.sup.SUB)NHR.sup.SUB (carbodiimide adduct),
C(.dbd.O)H (aldehyde), C(.dbd.O)R.sup.B (ketone), SH (sulfhydryl or
thiol), OH (alcohol), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl), R.sup.ALK--Hg--X (alkyl mercurial), and
S(.dbd.O).sub.2CR.sup.B.dbd.CR.sup.B.sub.2
(.alpha.,.beta.-unsaturated sulfone);
[0054] more preferably, each of the n.sup.[6] X p.sup.[6] moieties
shown as T.sup.[6] and each of the n.sup.[7].times.p.sup.[7]
moieties shown as T.sup.[7] is independently chosen from the group
NHR.sup.SUB (amine), C(.dbd.O)CH.sub.2X (haloacetyl), R.sup.ALKX
(alkyl halide), S(.dbd.O).sub.2OR.sup.ALKX (alkyl sulfonate),
NR.sup.1R.sup.2 wherein R.sup.1R.sup.2 is
--C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide),
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl), R X (alkyl mercurial), and
S(.dbd.O).sub.2CR.sup.B.dbd.CR.sup.B.sub.2
(.alpha.,.beta.-unsaturated sulfone);
[0055] even more preferably each of the n.sup.[6].times.p.sup.[6]
moieties shown as T.sup.[6] and each of the
n.sup.[7].times.p.sup.[7] moieties shown as T.sup.[7] is
independently chosen from the group NHR.sup.SUB (amine),
C(.dbd.O)CH.sub.2X (haloacetyl), NR.sup.1R.sup.2 wherein
R.sup.1R.sup.2 is --C(.dbd.O)CH.dbd.CHC(.dbd.O)-- (maleimide), and
C(.dbd.O)CR.sup.B.dbd.CR.sup.B.sub.2 (.alpha.,.beta.-unsaturated
carbonyl);
[0056] most preferably, all of the n.sup.[6].times.p.sup.[6]
moieties shown as T.sup.[6] and all of the
n.sup.[7].times.p.sup.[7] moieties shown as T.sup.[7] are
identical;
wherein
[0057] each X is independently a halogen of atomic number greater
than 16 and less than 54 or other good leaving group;
[0058] each R.sup.ALK is independently a linear, branched, or
cyclic alkyl (1-20C) group;
[0059] each R.sup.SUB is independently H, linear, branched, or
cyclic alkyl (1-20C), aryl (6-20C), or alkaryl (7-30C);
[0060] each R.sup.ESTER is independently N-succinimidyl,
p-nitrophenyl, pentafluorophenyl, tetrafluorophenyl,
pentachlorophenyl, 2,4,5-trichlorophenyl, 2,4-dinitrophenyl,
cyanomethyl and the like, or other activating group;
[0061] each R.sup.B is independently a radical comprising 1-50
atoms selected from the group C, H, N, O, Si, P and S;
[0062] n.sup.[6]=1 to 32, more preferably n.sup.[6]=1 to 16, even
more preferably n.sup.[6]=1 to 8, yet more preferably n.sup.[6]=1
to 4, most preferably n.sup.[6]=1 to 2;
[0063] p.sup.[6]=1 to 8, more preferably p.sup.[6]=1 to 4, most
preferably p.sup.[6]=1 to 2;
[0064] with the proviso that the product n.sup.[6].times.p.sup.[6]
be greater than 1 and less than 33;
[0065] n.sup.[7]=1 to 32, more preferably n.sup.[7]=1 to 16, even
more preferably n.sup.[7]=1 to 8, yet more preferably n.sup.[7]=1
to 4, most preferably n.sup.[7]=1 to 2;
[0066] p.sup.[7]=1 to 8, more preferably p.sup.[7]=1 to 4, most
preferably p.sup.[7]=1 to 2;
[0067] with the proviso that the product n.sup.[7].times.p.sup.[7]
be greater than 1 and less than 33;
[0068] each of the n.sup.[6] moieties shown as Q.sup.[6] and each
of the 2.times.n.sup.[7] moieties shown as Q.sup.[7] is
independently a radical comprising 1-100 atoms selected from the
group C, H, N, O, Si, P and S, containing attachment sites for at
least p.sup.[6] (for Q.sup.[6]) or p.sup.[7]/2 (for Q.sup.[7],
where p.sup.[7]/2 is an integer) functional groups on alkyl,
alkenyl, or aromatic carbon atoms;
[0069] more preferably, all of the n.sup.[6] moieties shown as
Q.sup.[6] are identical;
[0070] more preferably, all of the 2.times.n.sup.[7] moieties shown
as Q.sup.[7] are identical;
[0071] more preferably, each of the n.sup.[6] moieties shown as
Q.sup.[6], is independently chosen from the group
CH[(CH.sub.2).sub.r(attachment site)].sub.2 and
CH[(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q(attachment
site)].sub.2, wherein r=1 to 10 and q=1 to 10;
[0072] more preferably, each of the 2.times.n.sup.[7] moieties
shown as Q.sup.[7], is independently chosen from the group
(CH.sub.2).sub.r,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)(CH.sub.2).sub.q,
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.q,
(CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)
(CH.sub.2).sub.qNR.sup.SUBC(.dbd.O) (CH.sub.2).sub.r,
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2).sub.qNR.sup.SUBC(.dbd.O)
(CH.sub.2).sub.r, (CH.sub.2).sub.rNR.sup.SUBC(.dbd.O)
(CH.sub.2CH.sub.2O).sub.qCH.sub.2CH.sub.2, and
(CH.sub.2).sub.rC(.dbd.O)NR.sup.SUB(CH.sub.2CH.sub.2O).sub.qCH.sub.2CH.su-
b.2, wherein r=1 to 10, more preferably r=2 to 6, and q=1 to 10,
more preferably q=1 to 3.
BRIEF DESCRIPTION OF THE DRAWINGS
[0073] FIG. 1 shows the anti-PN response in mice primed with
PN-KLH, treated with [(PN).sub.20--BAHA]-EDDA, Conjugate 17-II, in
the doses shown or with saline, which were given a booster
injection of PN-KLH and then bled 5 days later. Sera were tested at
3 dilutions by the Farr assay using radiolabeled PN at 10.sup.-8 M
and the data are presented as the percentage reduction of anti-PN
antibodies. There were 5 mice per group.
[0074] FIG. 2 shows the anti-KLH response in mice primed with
PN-KLH, treated with [(PN).sub.20--BAHA]-EDDA, Conjugate 17-II, in
the doses shown, given a booster injection of PN-KLH and then bled
5 days later. Anti-KLH antibodies were assayed by enzyme-linked
immunosorbent assay (ELISA). The results are expressed as the
percent of a standard pool of antisera. There were 5 mice per
group.
[0075] FIG. 3 shows the anti-PN response in mice primed with
PN-KLH, treated with either [(PN).sub.16--BAHA.sub.ox]-EDDA
(Conjugate 11-IV), [(PN).sub.20--BAHA.sub.ox]-EDDA (Conjugate
11-II), [(PN).sub.24--BAHA.sub.ox]-EDDA (Conjugate 11-VI) or
[(PN).sub.32--BAHA.sub.ox]-EDDA (Conjugate 11-VIII) in the doses
shown, given a booster injection of PN-KLH and then bled 5 days
later. Sera were tested by the Farr assay using radiolabeled PN at
10.sup.-8 M. There were 5 mice per group.
[0076] FIG. 4 shows the anti-PN response in mice primed with
PN-KLH, treated with (PN).sub.20--HAD-AHAB-TEG, Conjugate 20-II, in
the doses shown or with HAD-AHAB only, or the PN only or a mixture
of each, then boosted with PN-KLH and bled 5 days later. Sera were
tested by the Farr assay using radiolabeled PN at a concentration
of 1-8 M. The percent reduction was calculated and the data are
presented. There were 5 mice per group.
[0077] FIG. 5 shows the anti-PN response in mice primed with
PN-KLH, treated with (PN).sub.20--HADPS-AHAB-TEG, Conjugate 20-IV,
in the doses shown, then boosted with PN-KLH and bled 5 days later.
Sera were tested by the Farr assay using radiolabeled PN at a
concentration of 10.sup.-8 M. There were 5 mice per group.
[0078] FIGS. 6A-C show the structure of the derivatized valency
platform molecule and the linker coupling the polynucleotide to the
platform molecule for Conjugates 3-I, 3-II, 11-I, 11-II, 11-IV,
11-VI, 11-VIII, 17-I, 17-II, 20-I, 20-II, 20-III, and 20-IV.
[0079] FIG. 7 shows the structures of the derivatized valency
platform molecule "HAD-AHAB-TEG."
[0080] FIG. 8 compares the level of T cell proliferation induced by
melittin peptides.
[0081] FIG. 9 compares the levels of anti-melittin peptide 2
antibodies produced in mice treated with melittin peptide Conjugate
2 versus the control mice treated with formulation buffer.
[0082] FIG. 10 compares the levels of anti-melittin antibodies
produced in mice treated with melittin peptide Conjugate 2 versus
the control mice treated with formulation buffer.
[0083] FIG. 11 compares the levels of anti-melittin peptide 2
antibody-forming cells in mice treated with melittin peptide
Conjugate 2 versus the control mice treated with formulation
buffer.
[0084] FIG. 12 illustrates that melittin peptide Conjugate 4, a
conjugate of peptide #5 which contains a T cell epitope, was not a
tolerogen.
[0085] FIG. 13 illustrates melittin conjugates within the present
invention.
[0086] FIG. 14 illustrates the increase in the percentage of
reduction in anti-dsPN antibody achieved by conjugates within the
present invention LJP-249A and LJP-249B which are Conjugate 3-II
compared to a prior art conjugate (LJP-105) comprising D-EK and
(PN).sub.50.
[0087] FIG. 15 illustrates the suppression of serum circulating IgG
anti-DNA antibodies in male BXSB mice treated with LJP-394,
Conjugate 20-II. An ELISA assay was used to measure IgG antibodies
to (PN).sub.50 conjugated to D-EK. The serum from each of eight
individual mice in each group was assayed.
MODES FOR CARRYING OUT THE INVENTION
[0088] As used herein "valency platform molecule" means a
chemically-defined, non-polymeric, nonimmunogenic molecule
containing sites which facilitate the attachment of a discreet
number of biological and/or chemical molecules.
[0089] "Nonimmunogenic" is used to describe the valency platform
molecule and means that the valency platform molecule elicits
substantially no immune response when it is administered by itself
or when administered as the platform portion of a conjugate to an
individual.
[0090] As used herein "individual" denotes a member of the
mammalian species and includes humans, primates, mice and domestic
animals such as cattle and sheep, sports animals such as horses,
and pets such as dogs and cats.
[0091] As used herein the term "immunogen" means a chemical entity
that elicits a humoral immune response when injected into an
animal. Immunogens have both B cell epitopes and T cell
epitopes.
[0092] The term "analog" of an immunogen intends a molecule that
(a) binds specifically to an antibody to which the immunogen binds
specifically and (b) lacks T cell epitopes. Although the analog
will normally be a fragment or derivative of the immunogen and thus
be of the same chemical class as the immunogen (e.g., the immunogen
is a polypeptide and the analog is a polypeptide), chemical
similarity is not essential. Accordingly, the analog may be of a
different chemical class than the immunogen (e.g., the immunogen is
a carbohydrate and the analog is a polypeptide) as long as it has
the functional characteristics (a) and (b) above. The analog may be
a protein, carbohydrate, lipid, lipoprotein, glycoprotein,
lipopolysaccharide, nucleic acid or other chemical or biochemical
entity.
[0093] An analog of an immunogen may also comprise a "mimotope."
The term "mimotope" intends a synthetic molecule which
competitively inhibits the antibody from binding the immunogen.
Because it specifically binds the antibody, the mimotope is
considered to mimic the antigenic determinants of the immunogen.
Like an analog of an immunogen, a mimotope (a) binds specifically
to an antibody to which the immunogen binds specifically and (b)
lacks T cell epitopes.
[0094] An analog of an immunogen may also comprise an "aptamer."
The term "aptamer" intends a synthetic oligonucleotide which
competitively inhibits the antibody from binding the immunogen.
Like an analog of an immunogen, an aptamer (a) binds specifically
to an antibody to which the immunogen binds specifically and (b)
lacks T cell epitopes.
[0095] As used herein the term "B cell anergy" intends
unresponsiveness of those B cells requiring T cell help to produce
and secrete antibody and includes, without limitation, clonal
deletion of immature and/or mature B cells and/or the inability of
B cells to produce antibody and/or apoptosis. "Unresponsiveness"
means a therapeutically effective reduction in the humoral response
to an immunogen. Quantitatively the reduction (as measured by
reduction in antibody production) is at least 50%, preferably at
least 75%, and most preferably 100%.
[0096] "Antibody" means those antibodies whose production is T cell
dependent.
[0097] The valency of a chemically-defined valency platform
molecule within the present invention can be predetermined by the
number of branching groups added to the platform molecule. Suitable
branching groups are typically derived from diamino acids,
triamines, and amino diacids. A conjugate within the instant
invention is biologically stabilized; that is, it exhibits an in
vivo excretion half-life of hours to days to months to confer
therapeutic efficacy. The chemically-defined valency platform
molecules of the instant invention are also substantially
nonimmunogenic (i.e., they exhibit no or only mild immunogenicity
when administered to animals), non-toxic at the doses given (i.e.,
they are sufficiently non-toxic to be useful as therapeutic agents)
and are preferably composed of a defined chemical structure. They
provide a non-immunogenic, non-toxic polyfunctional substrate to
which a multiplicity of biological or chemical molecules such as
polynucleotide duplexes may be attached covalently. They will
normally have an average molecular weight in the range of about 200
to about 200,000, usually about 200 to about 20,000, and are
homogeneous as compared to the prior art polymers which were a
mixture of compounds of widely fluctuating molecular weight.
Examples of particularly preferred, homogenous valency platform
molecules within the present invention are derivatized
2,2'-ethylenedioxydiethylamine (EDDA), triethylene glycol (TEG) and
polyethylene glycols (PEGs) having a molecular weight of about 200
to about 8,000.
[0098] Conjugation of a biological or synthetic molecule to the
chemically-defined platform molecule may be effected in any number
of ways, typically involving one or more crosslinking agents and
functional groups on the biological or synthetic molecule and
valency platform molecule.
[0099] The synthetic polynucleotide duplexes that are coupled to
the valency platform molecule are composed of at least about 20 bp
and preferably 20-50 bp. Polynucleotides described herein are
deoxyribonucleotides unless otherwise indicated and are set forth
in 5' to 3' orientation. Preferably the duplexes are substantially
homogeneous in length; that is, the variation in length in the
population will not normally exceed about .+-.20%, preferably
.+-.10%, of the average duplex length in base pairs. They are also
preferably substantially homogeneous in nucleotide composition;
that is, their base composition and sequence will not vary from
duplex to duplex more than about 10%. Most preferably they are
entirely homogeneous in nucleotide composition from duplex to
duplex.
[0100] Based on circular dichroic (CD) spectra interpretation, the
duplexes that are useful in the invention assume a B-DNA type
helical structure. It should be understood that it is not intended
that the invention be limited by this belief and that the duplexes
may, upon more conclusive analysis assume Z-DNA and/or A-DNA type
helical structures.
[0101] These polynucleotide duplexes may be synthesized from native
DNA or synthesized by chemical or recombinant techniques. Naturally
occurring or recombinantly produced dsDNA of longer length may be
digested (e.g., enzymatically, chemically or by mechanical
shearing) and fractionated (e.g., by agarose gel or Sephadex.RTM.
column) to obtain polynucleotides of the desired length.
[0102] Alternatively, pairs of complementary single-stranded
polynucleotide chains up to about 70 bases in length are readily
prepared using commercially available DNA synthesizers and then
annealed to form duplexes by conventional procedures. Synthetic
dsDNA of longer length may be obtained by enzymatic extension
(5'-phosphorylation followed by ligation) of the chemically
produced shorter chains.
[0103] The polynucleotides may also be made by molecular cloning.
For instance, polynucleotides of desired length and sequence are
synthesized as above. These polynucleotides may be designed to have
appropriate termini for ligation into specific restriction sites.
Multiple iterations of these oligomers may be ligated in tandem to
provide for multicopy replication. The resulting construct is
inserted into a standard cloning vector and the vector is
introduced into a suitable microorganism/cell by transformation.
Transformants are identified by standard markers and are grown
under conditions that favor DNA replication. The polynucleotides
may be isolated from the other DNA of the cell/microorganism by
treatment with restriction enzymes and conventional size
fractionation (e.g., agarose gel, Sephadex.RTM. column).
[0104] Alternatively, the polynucleotides may be replicated by the
polymerase chain reaction (PCR) technology. Saiki, R. K., et al.,
Science (1985) 230:1350; Sacki, et al., Science (1988) 239:487;
Sambrook, et al., In Molecular Cloning Techniques: A Laboratory
Manual, Vol. 12, p 14.1-14.35 Cold Spring Harbor Press (1989).
[0105] Polynucleotides may be screened for binding activity with
SLE antisera by the assays described in the examples. The modified
Farr assay in which binding activity may be expressed as I.sub.50
(the polynucleotide concentration in molar nucleotides resulting in
half-maximal inhibition) is a preferred assay. Polynucleotide
duplexes having an I.sub.50 of less than about 500 nM, preferably
less than 50 nM, are deemed to have significant binding activity
and are, therefore, useful for making the conjugates of this
invention.
[0106] The polynucleotides are conjugated to the chemically-defined
valency platform molecule in a manner that preserves their antibody
binding activity. This is done by conjugating the polynucleotide to
the valency platform molecule at a predetermined site on the
polynucleotide chain such that the polynucleotide forms a pendant
chain of at least about 20 base pairs measured from the conjugating
site to the free (unattached) end of the chain.
[0107] In a particularly preferred embodiment, the polynucleotides
of the invention conjugates are coupled to a linker molecule at or
proximate one of their ends. The linker molecule is then coupled to
the chemically-defined valency platform molecule. For example, a
defined double-stranded PN can be conjugated to a valency platform
molecule by first providing a single chain consisting of
approximately 20 alternating cytosine (C) and adenosine (A)
nucleotides. Four CA chains can then be covalently conjugated
through linkers such as HAD to four reactive sites on a derivatized
platform molecule such as triethylene glycol. The valency platform
molecule is synthesized to include groups such as bromoacetyl.
During the conjugation, a leaving group is displaced by sulfur. A
second single nucleotide chain consisting of approximately 20
alternating thymidine (T) and guanosine (G) nucleotides can then be
annealed to the CA strand to form a double-stranded PN conjugate of
the formula, [(PN).sub.20-linker].sub.4-valency platform
molecule.
[0108] Alternatively, in another preferred embodiment, the
polynucleotide may be coupled to the derivatized valency platform
molecule at the 3' end of the polynucleotide via a morpholino
bridge formed by condensing an oxidized 3' terminal ribose on one
of the strands of the polynucleotide with a free amino group on the
derivatized platform molecule and then subjecting the adduct to
reducing conditions to form the morpholino linkage. Such coupling
requires the derivatized platform molecule to have at least an
equal number of amino groups as the number of polynucleotide
duplexes to be bound to the platform molecule. The synthesis of
such a conjugate is carried out in two steps. The first step is
coupling one strand of the polynucleotide duplex to the derivatized
platform molecule via the condensation/reduction reaction described
above. The oxidized 3' terminal ribose is formed on the single
polynucleotide strand by treating the strand with periodate to
convert the 3' terminal ribose group to an oxidized ribose group.
The single-stranded polynucleotide is then added slowly to an
aqueous solution of the derivatized platform molecule with a pH of
about 6.0 to 8.0 at 2-8.degree. C. The molar ratio of
polynucleotide to platform molecule in all the conjugation
strategies will normally be in the range of about 2:1 to about
30:1, usually about 2:1 to about 8:1 and preferably about 4:1 to
6:1. In this regard, it is preferable that the conjugate not have
an excessively large molecular weight as large molecules,
particularly those with repeating units, of m.w. >200,000 may be
T-independent immunogens. See Dintzis et al., J. Immun. (1983)
131:2196 and J. Immun. (1989) 143:1239. During or after the
condensation reaction (normally a reaction time of 24 to 48 hr), a
strong reducing agent, such as sodium cyanoborohydride, is added to
form the morpholino group. The complementary strand of the duplex
is then added to the conjugate and the mixture is heated and slowly
cooled to cause the strands to anneal. The conjugate may be
purified by gel permeation chromatography.
[0109] An alternative to the ribose strategy is forming aldehyde
functionalities on the polynucleotides and using those
functionalities to couple the polynucleotide to the platform
molecule via reactive functional groups thereon. Advantage may be
taken of the fact that gem, vicinal diols, attached to the 3' or 5'
end of the polynucleotide, may be oxidized with sodium periodate to
yield aldehydes which can condense with functional amino groups of
the platform molecule. When the diols are in a ring system, e.g., a
five-membered ring, the resulting condensation product is a
heterocyclic ring containing nitrogen, e.g., a six-membered
morpholino or piperidino ring. The imino-condensation product is
stabilized by reduction with a suitable reducing agent; e.g.,
sodium borohydride or sodium cyanoborohydride. When the diol is
acyclic, the resulting oxidation product contains just one aldehyde
and the condensation product is a secondary amine.
[0110] Another procedure involves introducing alkylamino or
alkylsulfhydryl moieties into either the 3' or 5' ends of the
polynucleotide by appropriate nucleotide chemistry, e.g.,
phosphoramidite chemistry. The nucleophilic groups may then be used
to react with a large excess of homobifunctional cross-linking
reagent, e.g., dimethyl suberimidate, in the case of alkylamine
derivatives, or an excess of heterobifunctional cross-linking
reagent, e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS)
or succinimidyl (4-iodoacetyl)aminobenzoate (SIAB), for the
alkylsulfhydryl derivatives. Once excess cross-linker is removed,
the polynucleotide derivatives are reacted with amino groups on the
platform molecule. Alternatively, the sulfhydryl group may be
reacted with an electrophilic center on the platform, such as a
maleimide or .alpha.-haloacetyl group or other appropriate Michael
acceptor.
[0111] Still another strategy employs modified nucleosides.
Suitable deoxynucleoside derivatives can be incorporated, by
standard DNA synthetic chemistry, at desired positions in the
polynucleotide, preferably on the 5' or 3' ends. These nucleoside
derivatives may then react specifically and directly with
alkylamino groups on the platform molecule. Alternatively, side
reactions seen with the above-described dialdehyde chemistry, such
as amine catalyzed beta-elimination, can be circumvented by
employing appropriate nucleoside derivatives as the 3' terminus of
the chain to be attached. An example of this is 5' methylene
extension of ribose; i.e., a 5' (2-hydroxyethyl)-group instead of a
5' hydroxymethyl group. An alternative would be to use a
phosphonate or phosphinate linkage for the 3' terminal dinucleotide
of the polynucleotide to be attached to the platform molecule.
Analogs of Immunogens
[0112] Immunogens that are involved in antibody-mediated
pathologies may be external (foreign to the individual) immunogens
such as allergens, sperm associated with male infertility, the
rheumatic fever carbohydrate complex, the RBC Rh/D antigen
associated with hemolytic disease of the newborn, biological drugs,
including native biological substances foreign to the individual
such as therapeutic proteins, peptides and antibodies, and the like
or self-immunogens (autoimmunogens) such as those associated with
thyroiditis (thyroglobulin), stroke (cardiolipin) and myasthenia
gravis (acetylcholine receptor).
[0113] Analogs to such immunogens may be identified by screening
candidate molecules to determine whether they (a) bind specifically
to serum antibodies to the immunogen and (b) lack T cell epitopes.
Specific binding to serum antibodies may be determined using
conventional immunoassays and the presence or absence of T cell
epitopes may be determined by conventional T cell activation
assays. In this regard, an analog which "binds specifically" to
serum antibodies to the immunogen exhibits a reasonable affinity
thereto. Further in this regard, it should be recognized that
testing for T cell epitopes is conducted on a subject-by-subject
basis using T cells taken from an intended recipient or from
various patients that represent the target population of
recipients. The presence or absence of T cell epitopes may be
determined using the tritiated thymidine incorporation assay
described in the examples. The presence of T cell epitopes can also
be determined by measuring secretion of T cell-derived lymphokines
by methods well known in the art. Analogs that fail to induce
statistically significant incorporation of thymidine above
background are deemed to lack T cell epitopes. It will be
appreciated that the quantitative amount of thymidine incorporation
may vary with the immunogen. Typically a stimulation index below
about 2-3, more usually about 1-2, is indicative of a lack of T
cell epitopes.
[0114] A normal first step in identifying useful analogs is to
prepare a panel or library of candidates to screen. For instance,
in the case of protein or peptide analogs, libraries may be made by
synthetic or recombinant techniques such as those described by
Geysen et al. in Synthetic Peptides as Antigens; Ciba Symposium
(1986) 119:131-149; Devlin et al., Science (1990) 249:404-406;
Scott et al., Science (1990) 249:386-390; and Cwirla et al., PNAS
USA (1990) 87:6378-6382. In one synthetic technique, peptides of
about 5 to 30 amino acids are synthesized in such a manner that
each peptide overlaps the next and all linear epitopes are
represented. This is accomplished by overlapping both the carboxyl
and amino termini by one less residue than that expected for a B
cell epitope. For example, if the assumed minimum requirement for a
B cell epitope is six amino acids, then each peptide must overlap
the neighboring peptides by five amino acids. In this embodiment,
each peptide is then screened against antisera produced against the
native immunogen, either by immunization of animals or from
patients, to identify the presence of B cell epitopes. Those
molecules with antibody binding activity are then screened for the
presence of T cell epitopes as described in the examples. The
molecules lacking T cell epitopes are useful as analogs in the
invention.
[0115] If the T cell epitope(s) of an immunogen are known or can be
identified, random T cell screening of candidate analogs is not
necessary. In such instances, the T cell epitope(s) may be altered
(e.g., by chemical derivatization, or elimination of one or more
components of the epitope) to render them inoperative or be
eliminated completely, such as, for instance, in the case of
peptides, by synthetic or recombinant procedures.
[0116] Mimotopes and aptamers are synthesized by conventional
methods and are screened in the same manner as other analogs of
immunogens.
[0117] The analogs are coupled to a nonimmunogenic valency platform
molecule to prepare the conjugates of the invention. Conjugates
comprising valency platform molecules and biologically active
molecules such as carbohydrates, lipids, lipopolysaccharides,
proteins, glycoproteins, drugs, and analogs of interest are
synthesized utilizing the chemistries exemplified herein. A
preferred method of synthesis is to incorporate a linker molecule
on the biological molecule by well known methods chosen on a
case-by-case basis.
[0118] When conjugating drugs such as adriamycin (doxorubicin) to a
valency platform molecule, the amino group on a sugar ring can
react with platform molecules containing active esters. Adriamycin
can also be modified to contain thiol groups for conjugation to a
haloacetylated platform (Kaneko, T., et al., Bioconjugate
Chemistry, 2:133 (1991)).
[0119] Carbohydrates such as oligosaccharides can be modified to
contain a sulfhydryl-containing linker (Wood, S. J. and Wetzel, R.,
Bioconjugate Chemistry, 3:391 (1992)). The sulfhydryl group is used
for conjugation to a haloacetylated platform. Alternatively,
carbohydrates can be oxidized to generate aldehydes which are
reacted with amino platforms in the presence of NaCNBH.sub.3 to
form conjugates.
[0120] Lipids such as glycolipids containing an ethanolamine group
are reacted with an activated carboxylate on the platform.
Lipopolysaccharides containing sugar units are oxidized to generate
aldehydes which are reacted in the presence of NaCNBH.sub.3 with
amino platforms to form conjugates by reductive amination.
[0121] In the case of additional proteins such as Fab' antibody
fragments, sulfhydryl groups on the protein (Fab') are conjugated
to a platform via haloacetyl groups. Glycoproteins are modified
with a thiol linker using iminothiolate. The thiol reacts with
platforms containing haloacetyl groups.
[0122] The ability of the conjugates to act as tolerogens and
specifically suppress production of antibodies may be evaluated in
the murine model described in the examples.
[0123] The conjugates will normally be formulated for
administration by injection, (e.g., intraperitoneally,
intramuscularly, intravenously etc.). Accordingly, they will
typically be combined with pharmaceutically acceptable aqueous
carriers such as saline, Ringer's solution, dextrose solution, and
the like. The conjugate will normally constitute about 0.01% to 10%
by weight of the formulation. The conjugate is administered to an
individual in amounts sufficient to at least partially reestablish
tolerance to the autoantigens causing SLE. Such amounts are
sometimes herein referred to as "therapeutically effective"
amounts. The particular dosage regimen i.e., dose, timing and
repetition, will depend upon the particular individual, and that
individual's medical history. Normally a dose of about 1 to 1000
.mu.g conjugate/kg body weight will be given. Repetitive
administrations may be required to achieve and/or maintain a state
of immune tolerance.
[0124] The following examples further illustrate the invention and
its unexpectedness relative to the prior art. These examples are
not intended to limit the invention in any manner.
EXAMPLE 1
[0125] The following reaction schemes illustrate methods of
synthesizing derivatized chemically-defined valency platform
molecules within the present invention. In this example,
DMTr=4,4'-dimethoxytriphenylmethyl; Tr=trityl; Bz=benzoyl;
Cp=deoxycytidine monophosphate, CE=cyanoethyl; CPG=controlled pore
glass, DMF=dimethylformamide, DCC=dicyclohexylcarbodiimide,
TFA=trifluoroacetic acid, CDI=carbonyl diimidazole, Ts=tosyl
(para-toluene sulfonyl), DIPAT=diisopropyl ammonium tetraazolide,
TBDMSCl=tertbutyl dimethyl silyl chloride, TBAF=tetrabutyl ammonium
fluoride, NMMO=N-methylmorpholine-N-oxide.
##STR00001##
##STR00002## ##STR00003##
##STR00004##
##STR00005##
##STR00006##
##STR00007##
##STR00008##
##STR00009##
##STR00010##
##STR00011##
[0126] Synthesis of reagents used to modify (CA).sub.8,
(CA).sub.10, (CA).sub.12 and (CA).sub.16 with disulfide linkers is
described in Reaction Scheme 11 below:
##STR00012##
[0127] Synthesis of a reagent used to modify (CA).sub.25 with
vicinal diol linkers is described in Reaction Scheme 12 below:
##STR00013##
##STR00014##
EXAMPLE 2
Synthesis of Chemically-Defined Valency Platform Molecules
[0128] Compound 1-[3,5-Bis-(iodoacetamido) benzoic acid]: 2.93 g
(8.28 mmol, 2.2 eq) of iodoacetic anhydride was added to a stirred
suspension of 572 mg (3.76 mmol) of 3,5-diaminobenzoic acid in 19
mL of dioxane at room temperature under N.sub.2 atmosphere. The
mixture was stirred, covered with foil for 20 hours and partitioned
between 50 mL of EtOAc and 50 mL of 1N HCl solution. The EtOAc
layer was washed with brine, dried over MgSO.sub.4, filtered, and
concentrated on a rotary evaporator to give 3.3 g of tan solid. The
material was purified by silica gel chromatography (94/5/1
CH.sub.2Cl.sub.2/MeOH/HOAc) to yield 992 mg (54%) of compound 1 as
a white solid: NMR (DMSO) 3.84 (s, 4H), 7.91 (s, 2H), 8.14 (s, 1H),
10.56 (s, 2H).
[0129] Compound 2--[3,5-Bis-(iodoacetamido) benzoyl chloride]: 117
.mu.L (1.6 mmol, 190 mg) of SOCl.sub.2 was added to a solution of
390 mg (0.8 mmol) of 1 in 34 mL of THF. The mixture was refluxed
under N.sub.2 atmosphere until all solids had dissolved
(approximately 30 minutes) to give a clear red-brown solution. The
mixture was concentrated on the rotary evaporator and placed under
vacuum to provide crude compound 2 as a foamy solid which was used
directly in the next step.
[0130] Compound 3--[N,N'-Bis-(3,5-bis-(iodoacetamido) benzoyl)
derivative of .alpha.,.omega.-bis-(N-2-aminoethylcarbamoyl)
polyethyleneglycol]: 570 mg of
.alpha.,.omega.-bis-(N-2-aminoethylcarbamoyl) polyethyleneglycol
(0.16 mmol, 3350 g/mol, Sigma) was placed in a tared flask. Toluene
(20 mL) was added and water was removed by azeotropic distillation.
The residue was dried under vacuum to give 549 mg of solid and
dissolved in 4 mL THF with 89 .mu.L (0.64 mmol) of
diisopropylethylamine. The crude acid chloride was dissolved in 4
mL anhydrous THF and added to the mixture over 30 seconds under
N.sub.2. The mixture was stirred for 16 hours at room temperature
and partitioned between 25 mL of 0.1 N HCl and 25 mL of
CH.sub.2Cl.sub.2. The aqueous layer was again extracted with
CH.sub.2Cl.sub.2 and the organic layers were combined, washed with
25 mL of H.sub.2O, followed by 50 mL of at NaHCO.sub.3 solution.
The organic layers were dried with Na.sub.2SO.sub.4, filtered, and
concentrated to give 784 mg of orange oil. Silica gel
chromatography (9/1 CH.sub.2Cl.sub.2/MeOH) yielded 190 mg of
colorless oil which was crystallized from hot EtOH/Et.sub.2O,
collected on sintered glass filter under N.sub.2 pressure, and
dried under vacuum to provide 177 mg of compound 3 as a white
solid: NMR (CDCl.sub.3) 3.40 (bd m, 8H), 3.59 (bd s,
(CH.sub.2CH.sub.2O).sub.n, integral too large to integrate in
relation to other integrals), 3.91 (s, 8H), 4.21 (m, 4H), 6.04 (bd
m, 2H), 7.55 (bd m, 2H), 7.78 (bd s, 4H), 8.10 (bd s, 2H), 9.30 (bd
m, 4H): iodoacetyl determination (European Journal of Biochemistry
(1984) 140:63-71): Calculated, 0.92 mmol/g; Found, 0.96 mmol/g.
[0131] Compound
4--[Mono-N-carbobenzyloxy-3,6-dioxa-1,8-diaminooctane]: A solution
of 14.3 mL (17.1 g, 100 mmol) of benzylchloroformate in 200 mL of
CH.sub.2Cl.sub.2 was added dropwise over a 1 hour period to a
solution of 29.0 mL (29.6 g, 200 mmol) of
1,2-bis-(2'-aminoethoxy)ethane (Fluka) in 100 mL of
CH.sub.2Cl.sub.2 at 0.degree.. The mixture was stirred at room
temperature for 24 hours and 1 N HCl was added until the aqueous
layer remained acidic (pH less than 2). The aqueous layer was
washed with three 50 mL portions of CH.sub.2Cl.sub.2 and
neutralized with 1 N NaOH until the pH was above 13. The basic
aqueous layer was extracted with five 75 mL portions of
CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers were dried
(MgSO.sub.4), filtered, and concentrated to yield 12.7 g (45%) of
compound 4 as a thick oil: .sup.1H NMR (CDCl.sub.3) .delta. 2.82
(bd s, 2H), 3.30-3.60 (m, 12H), 5.10 (s, 2H), 5.75 (bd s, 1H),
7.20-7.40 (m, 5H); .sup.13C NMR (CDCl.sub.3) .delta. 41.1, 41.8,
66.5, 70.0, 70.2, 70.4, 73.5, 127.9, 128.0, 128.4, 136.9,
156.4.
[0132] Compound 5--[N-tert-butyloxycarbonyliminodiacetic acid]:
This compound was prepared by a procedure similar to that reported
by Garrigues, B. and Lazraq, E. M. Tetrahedron Letters (1986) 27,
1685-1686. 47 mL (34.2 g, 338 mmol) of Et.sub.3N was added to a
stirred solution of 22.0 g (169 mmol) of iminodiacetic acid and
36.8 g (169 mmol) of di-tert butyldicarbonate in 169 mL of 50/50
dioxane/H.sub.2O at room temperature. The mixture was stirred for
24 hours and most of the dioxane was removed on a rotary
evaporator. The mixture was partitioned between 350 mL of 1 N HCl
and five 100 mL portions of EtOAc. The combined EtOAc layers were
dried (MgSO.sub.4), filtered, and concentrated to give a white
solid. Recrystallization from hexanes/EtOAc yielded 35.3 g (90%) of
compound 5 as crystals: m.p. 131-132.degree. fused; .sup.1H NMR
(DMSO) d 1.35 (s, 9H), 3.87 (s, 2H), 3.91 (s, 2H), 12.6 (bd s, 2H);
.sup.13C NMR (DMSO) d 27.9, 49.6, 79.6, 154.8, 171.2.
[0133] Compound 6. 9.99 g (48.5 mmol) of dicyclohexylcarbodiimide
was added to a solution of 4.52 g 73 (19.4 mmol) of compound 5 and
4.46 g (38.8 mmol) of N-hydroxysuccinimide in 100 ML of THF at
0.degree.. The mixture was stirred for 3 hours at 0.degree. C., and
a solution of 5.39 mL (3.92 g, 38.8 mmol) Et.sub.3N and 10.9 g
(38.7 mmol) of compound 4 in 83 mL of THF was added, and the
mixture was stirred at 5.degree. C. for 17 hours. The mixture was
filtered to remove solids, and the filtrate was concentrated to an
oil which was partitioned between 400 mL of EtOAc and two 100 mL
portions of 1 N HCl. The EtOAc layer was washed with three, 100 mL
portions of 1 N Na.sub.2CO.sub.3, 100 mL of brine, dried
(MgSO.sub.4), filtered and concentrated to provide 14.2 g (96%) of
compound 6 as a thick oil; .sup.1H NMR (CDCl.sub.3) .delta. 1.41
(s, 9H), 3.30-3.70 (m, 24H), 3.70-3.90 (m, 4H), 5.10 (s, 4H), 5.50
(bd s, 2H), 7.12 (bd s, 1H), 7.30-7.40 (m, 10H), 8.24 (bd s,
1H).
[0134] Compound 7. 26.3 mL (38.9 g, 156 mmol) of trifluoroacetic
acid was added to a solution of 14.2 g (18.6 mmol) of compound 6 in
111 mL of CH.sub.2Cl.sub.2 and the mixture was stirred at room
temperature for 3 hours. The mixture was concentrated on the rotary
evaporator to give a viscous oil, and the oil was dissolved in 93
mL of THF. The solution was cooled to 0.degree. C. and 3.72 g (37.2
mmol) of succinic anhydride was added followed by 5.18 mL (3.76 g,
37.2 mmol) of Et.sub.3N. The cooling bath was removed, and the
mixture was stirred for 18 hours at room temperature. The solvent
was removed under reduced pressure, and the resulting oil was
partitioned between 300 mL of CH.sub.2Cl.sub.2 and three 100 mL
portions of H.sub.2O. The CH.sub.2Cl.sub.2 layer was dried
(MgSO.sub.4), filtered, and concentrated to provide an oil which
was purified by chromatography on silica gel (9/1/0.1
EtOAc/MeOH/acetic acid) to provide 10.5 g (74%) of compound 7 as a
viscous oil; .sup.1H NMR (CDCl.sub.3) .delta. 2.50-2.60 (m, 4H),
3.30-3.60 (m, 24H), 3.88 (s, 2H), 4.03 (s, 2H), 5.07 (s, 4H), 5.77
(bd s, 2H), 7.20-7.30 (m 10H), 7.91 (bd s, 2H), 8.88 (bd s, 1H);
.sup.13C(CDCl.sub.3) .delta. 27.7, 29.0, 39.4, 41.0, 52.9, 53.8,
66.5, 69.3, 69.8, 70.0, 70.1, 127.8, 128.1, 128.3, 136.7, 156.6,
169.1, 169.6, 173.3, 174.5.
[0135] Compound 8--[4-Nitrophenyl ester of compound 7]: 1.61 g
(7.83 mmol) of dicyclohexylcarbodiimide was added to a solution of
3.98 g (5.22 mmol) of 7 and 800 mg (5.75 mmol) of 4-nitrophenol in
26 mL of CH.sub.2Cl.sub.2 at 0.degree.. The mixture was stirred at
room temperature under N.sub.2 for 64 hours. The mixture was cooled
to 0.degree., 1 mL of HOAc was added, and the mixture was kept at
0.degree. for 2 hours. The solids were removed by filtration, and
the filtrate was concentrated. The residue was purified by silica
gel chromatography (gradient, 91/8/1 to 84/15/1
CH.sub.2Cl.sub.2/IPA/HOAc) to provide 2.58 g (56%) of compound 8 as
a viscous oil: .sup.1H NMR (CDCl.sub.3) .delta. 2.66 (t, 2H), 2.84
(t, 2H), 3.32-3.68 (m, 24H), 3.90 (bd s, 2H), 4.01 (bd s, 2H), 5.06
(s, 4H), 5.58 (bd m, 2H), 6.91 (bd m, 1H), 7.27 (d, 2H), 7.33 (s,
10H), 8.23 (d, 2H), 9.01 (bd m, 1H).
[0136] Compound 10--[4-Nitrophenylbromoacetate]: 9.28 g (45 mmol)
of dicyclohexylcarbodiimide was added to a stirred solution of 5.0
g (35.9 mmol) of bromoacetic acid and 8.50 g (61.1 mmol) of
4-nitrophenol in 180 mL of EtOAc at 0.degree.. The mixture was
stirred for 16 hours at 5.degree. and 1 mL of acetic acid was
added. The mixture was stirred for 20 minutes at room temperature
and then placed in a freezer for 20 minutes. The solid material was
removed by filtration, and the filtrate was concentrated to a
viscous oil and crystallized from Et.sub.2O/hexanes to provide 7.73
g (83%) of compound 10 as flakes: m.p. 86-87.degree.; TLC Rf=0.63
(50/50/1 hexanes/EtOAc/HOAc); .sup.1H NMR (CDCl.sub.3) .delta. 4.13
(s, 2H), 7.36 (d, J=12 Hz, 2H), 8.32 (d, J=12 Hz, 2H); .sup.13C NMR
(CDCl.sub.3) .delta. 24.9, 122.1, 125.3, 155.5 164.9; Anal. calc'd
for C.sub.8H.sub.6BrNO.sub.4: C, 36.95; H, 2.33; N, 5.39. Found: C,
37.24; H, 2.33; N, 5.42.
[0137] Compound 9: 300 mg (3.57 mmol) of NaHCO.sub.3, followed by
162 mg (1.09 mmol) of 2,2.sub.1-(ethylenedioxy)-diethylamine
(Fluka), was added to a solution of 2.37 g (2.68 mmol) of compound
8 in 15 mL of dioxane and 8 mL of H.sub.2O. The mixture was stirred
for 24 hours at room temperature and concentrated under vacuum to
approximately one half the original volume. The concentrate was
partitioned between 40 mL of CH.sub.2Cl.sub.2 and 40 mL of
saturated NaHCO.sub.3 solution. The CH.sub.2Cl.sub.2 layer was then
washed twice with 40 mL of 0.5 N HCl. The CH.sub.2Cl.sub.2 layer
was washed with saturated NaCl solution, dried (MgSO.sub.4),
filtered, and concentrated to give 2.8 g of an oil. This crude
produce was purified by silica gel chromatography (3/6/1
CH.sub.2Cl.sub.2/THF/MeOH) to provide 940 mg (59%) of compound 9 as
an oil: TLC R.sub.f=0.21 (3/6/1 CH.sub.2Cl.sub.2/THF/MeOH); .sup.1H
NMR (CDCl.sub.3) .delta. 2.45 (m, 4H), 2.59 (m, 4H), 3.25-3.55 (m,
60H), 3.87 (s, 4H), 4.05 (s, 4H), 5.07 (s, 8H), 5.62 (bd s, 4H),
6.78 (bd s, 2H), 7.34 (bd s, 20H), 8.56 (bd s, 2H); .sup.13C NMR
(CDCl.sub.3) .delta. 28.1, 30.3, 31.1, 39.4, 41.1, 52.9, 53.9,
66.5, 69.4, 69.7, 69.9, 70.2, 125.3, 127.8, 128.3, 136.8, 156.5,
168.8, 169.4, 172.1, 173.5.
[0138] Compound 34: 110 mg of 10% Pd on carbon was added to a
solution of 281 mg (0.175 mmol) of compound 9 in 5 mL of EtOH and 2
mL of cyclohexene under nitrogen and the resulting mixture was
refluxed under nitrogen for 2 hours. When cool, the mixture was
filtered through diatomaceous earth and concentrated under vacuum
to give 170 mg (92%) of compound 34 as an oil which was used
directly in the next step without purification; .sup.1H NMR
(CDCl.sub.3) .delta. 2.45 (m, 4H), 2.53 (m, 4H), 2.62 (m, 4H), 2.86
(m, 8H), 3.42-3.60 (m, 52H), 4.00 (s, 4H), 4.14 (s, 4H); .sup.13C
NMR (CDCl.sub.3) .delta. 28.2, 30.3, 31.1, 39.4, 41.1, 46.5, 48.6,
52.9, 53.8, 69.4, 69.7, 70.2, 72.4, 168.9, 169.5, 172.3, 173.8.
[0139] Compound 11: 128 mg (1.4 mmol) of NaHCO.sub.3 and 200 mg
(0.85 mmol) of compound 10 were added to a solution of 165 mg
(0.155 mmol) of compound 34 in 6 mL of dioxane and 3 mL of
H.sub.2O. The resulting mixture was stirred for 24 hours at room
temperature and concentrated under vacuum. The concentrate was
purified by chromatography on Sephadex.RTM. G-10 (MeOH) to give 114
mg (46%) of compound 11 as a viscous oil. An analytical sample was
prepared by preparative HPLC (C.sub.18; gradient 15/85/0.1 to
30/70/0.1 CH.sub.3CN/H.sub.2O/CF.sub.3CO.sub.2H, 50 min, 225 nm):
.sup.1H NMR (CDCl.sub.3) .delta. 2.58 (m, 4H), 2.65 (m, 4H),
3.43-3.62 (m, 60H), 3.92, (s, 8H), 4.03 (s, 4H), 4.16 (s, 4H); MS
(FAB) m/e (relative intensity) MNa+ 1605 (100), MH+ 1579 (1), 1581
(5), 1583 (7), 1585 (6), 1587 (2).
[0140] Compound 12--[Mono-N-carbobenzyloxy-1,6-diaminohexane]: A
solution of 21 mL (25.7 g, 150 mmol) of benzylchloroformate in 200
mL of dioxane was added dropwise to a stirred solution of 17.49 g
(150 mmol) of 1,6-hexanediamine and 19.58 g (196 mmol) of
KHCO.sub.3 in 100 mL of dioxane and 300 mL of H.sub.2O at
0.degree.. The mixture was stirred at room temperature for 18 hours
and then cooled to 0.degree.. The mixture was acidified with 12 N
HCl and extracted with two 100 mL portions of Et.sub.2O. The
aqueous layer was neutralized with 10 N NaOH and extracted with
eight 100 mL portions of Et.sub.2O. The basic extracts were
combined, dried (Na.sub.2SO.sub.4), and concentrated to provide
5.03 g (13%) of crude compound 12 as a semisolid residue: .sup.1H
NMR (DMSO) d 1.22-1.51 (m, 8H), 2.54 (t, 2H), 3.02 (d of t, 2H),
5.05 (s, 2H), 7.30-7.48 (m, 5H).
[0141] Compound 13: 918 mg (4.45 mmol) of dicyclohexylcarbodiimide
was added to a solution of 417 mg (1.78 mmol) of compound 5 and 409
mg (3.56 mmol) of NHS in 15 mL of THF at 0.degree.. The mixture was
stirred at 0.degree. for 4.5 hours and a solution of 1.02 g (4.08
mmol) of compound 12 in 4 mL of THF was added. The mixture was
stirred under N.sub.2 at 5.degree. for 18 hours. The concentrate
was partitioned between 30 mL of EtOAc and two 30 mL portions of 1
N HCl. The combined EtOAc layers were washed successively with 30
mL of H.sub.2O and 30 mL of saturated NaHCO.sub.3 solution, dried
(MgSO.sub.4), filtered, and concentrated to provide 1.48 g of
viscous residue. Purification by chromatography on silica gel (5/95
MeOH/CH.sub.2Cl.sub.2) gave 1.04 g (84%) of compound 13 as a sticky
solid: .sup.1H NMR (CDCl.sub.3) .delta. 1.33 (m, 8H), 1.43 (s, 9H),
1.51 (m, 8H), 3.18 (m, 4H), 3.26 (m, 4H), 3.81 (s, 2H), 3.85 (s,
2H), 4.90 (bd s, 2H), 5.10 (s, 4H), 6.81 (bd s, 1H), 7.28-7.40 (m,
10H), 8.05 (bd s, 1H).
[0142] Compound 14: 14.9 mL of trifluoroacetic acid was added to a
solution of 5.16 g (7.45 mmol) of compound 13 in 14.9 mL of
CH.sub.2Cl.sub.2 and the resulting mixture was stirred for 3 hours
at room temperature. The mixture was concentrated under vacuum and
redissolved in 57 mL of THF. 2.07 mL (1.51 g, 14.9 mmol) of
Et.sub.3N was added to the mixture. 1.5 g (14.9 mmol) of succinic
anhydride was added to the mixture and the mixture was then stirred
for 18 hours. The mixture was partitioned between 75 mL of 1 N HCl
and four 75 mL portions of CH.sub.2Cl.sub.2. The combined
CH.sub.2Cl.sub.2 layers were dried (MgSO.sub.4), filtered, and
concentrated to provide a solid. Crystallization from
CH.sub.2Cl.sub.2/EtOAc/hexanes provided 3.84 g (74%) of compound 14
as a white solid: m.p. 122.degree.; .sup.1H NMR (MeoH) d 1.32 (m,
8H), 1.48 (m, 8H), 2.56 (m, 4H), 3.10 (t, 4H), 3.23 (m, 4H), 4.00
(s, 2H), 4.18 (s, 2H), 5.05 (s, 4H), 7.33 (m, 10H).
[0143] Compound 15--[4-Nitrophenyl ester of compound 14]: 887 mg
(4.30 mmol) of dicyclohexylcarbodiimideo was added to a solution of
2.0 g (2.87 mmol) of compound 14 and 438 mg (3.15 mmol) of
4-nitrophenol in 15 mL of THF at 0.degree.. The mixture was allowed
to come to room temperature, stirred for 18 hours, and then cooled
to 0.degree.. 200 uL of acetic acid was then added and the mixture
was stirred at 0.degree. for 1 hour. The solids were removed by
filtration and the filtrate was concentrated to an oil.
Purification by chromatography on silica gel (92/8
CH.sub.2Cl.sub.2/IPA) and recrystallization of the resulting solid
from CH.sub.2Cl.sub.2/hexanes provided 1.52 g (64%) of compound 15
as a white solid: m.p. 65-68.degree.; .sup.1H NMR (CDCl.sub.3)
.delta. 1.30 (m, 8H), 1.47 (m, 8H), 2.71 (t, 2H), 2.90 (t, 2H),
3.17 (m, 4H), 3.25 (m, 4H), 3.92 (s, 2H), 4.08 (s, 2H), 4.86 (bd t,
1H), 4.95 (bd t, 1H), 5.09 (s, 4H), 6.28 (bd t, 1H), 7.23 (d, J=9
Hz, 2H), 7.32 (m, 10H), 8.22 (d, J=9 Hz, 2H), 8.95 (bd t, 1H).
[0144] Compound 16: A solution of 830 mg (0.99 mmol) of compound 15
in 7.5 mL of dioxane was added to a solution of 58 uL (59 mg, 0.40
mmol) of 2,2'-(ethylenedioxy)-diethylamine (Fluka) and 111 mg (1.31
mmol) of NaHCO.sub.3 in 7.5 mL of H.sub.2O. The mixture was stirred
at room temperature for 18 hours. The mixture was partitioned
between 50 mL of 1 N HCl and 50 mL of CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layer was dried (Na.sub.2SO.sub.4), filtered, and
concentrated to provide 1.28 g of viscous oil. Purification by
silica gel chromatography (84/15/1 CH.sub.2Cl.sub.2/MeOH/HOAc) gave
670 mg of compound 16 as a waxy solid: .sup.1H NMR (CDCl.sub.3)
.delta. 1.32 (m, 16H), 1.49 (m, 16H), 2.46 (m, 4H), 2.58 (m, 4H),
3.10-3.23 (m, 16H), 3.34 (m, 4H) 3.48 (m, 4H), 3.53 (s, 4H), 3.85
(s, 4H), 4.02 (s, 4H), 5.05 (s, 8H), 5.07 (underlying bd t, 2H),
5.15 (bd t, 2H), 7.30 (m, 20H), 7.40 (bd t, 2H), 8.60 (bd t,
2H).
[0145] Compound 35: A solution of 613 mg (0.41 mmol) of compound 16
in 20.3 mL of EtOH and 10.1 mL of cyclohexene was stirred
and-purged with nitrogen. 20 mg of 10% Pd on carbon (Aldrich) was
added and the mixture was heated in a 85.degree. oil bath for 1.5
hours. When cool, the mixture was filtered through diatomaceous
earth using 50/50H.sub.2O/acetone to rinse the flask and filter.
The filtrate was concentrated under vacuum to give 448 mg (114%) of
compound 35 as a waxy solid: .sup.1H NMR (D.sub.2O) .delta. 1.39
(m, 16H), 1.59 (m, 16H), 2.57 (t, 4H), 2.65 (t, 4H), 2.88 (t, 8H),
3.23 (t, 4H), 3.29 (t, 4H), 3.42 (t, 4H), 3.65 (t, 4H), 3.71 (s,
4H), 4.06 (s, 4H), 4.30 (s, 4H).
[0146] Compound 17: 546 mg (6.50 mmol) of NaHCO.sub.3 was added to
a solution of 445 mg (0.406 mmol) of compound 35 in 9.5 mL of
H.sub.2O. A solution of 838 mg (3.25 mmol) of compound 10 in 14.4
mL of dioxane was added to the resulting mixture. The mixture was
stirred for 7 hours at room temperature and partitioned between 50
mL of 0.1 N H.sub.2SO.sub.4 and 50 mL of CH.sub.2Cl.sub.2. The
CH.sub.2Cl.sub.2 layer was discarded, and the aqueous layer was
extracted with two 50 mL portions of CH.sub.2Cl.sub.2, two 50 mL
portions of 9/1 CH.sub.2Cl.sub.2/MeOH, 50 mL of 4/1
CH.sub.2Cl.sub.2/MeOH, and 50 mL of 3/2 CH.sub.2Cl.sub.2/MeOH. The
extracts were combined and dried (Na.sub.2SO.sub.4), filtered, and
concentrated to provide 282 mg of solid. Crystallization from
EtOH/EtOAc/Et.sub.2O gave 143 mg (24%) of compound 17 as a white
solid: .sup.1H NMR (CDCl.sub.3/MeOH) d 1.33 (m, 16H), 1.55 (m,
16H), 2.55 (m, 8H), 3.21 (m, 16H), 3.39 (m, 4H), 3.55 (m, 4H), 3.81
(s, 8H), 3.95 (s, 4H), 4.12 (s, 4H). Anal. calc'd for
C.sub.54H.sub.94N12O.sub.14Br.sub.4: C, 44.57; H, 6.51; N, 11.55;
Br, 21.97. Found: C, 45.85; H, 6.49; N, 11.37; Br, 19.90.
[0147] Compound
18--[1,5-Bis(N-carbobenzyloxy-6-aminohexanoamido)-3-azapentane]:
3.09 g (19.0 mmol) of carbonyldiimidazole was added to a solution
of 5.05 g (19.0 mmol) of N-carbobenzyloxy-6-aminohexanoic acid in
25 mL of EtOAc at room temperature. The mixture was stirred for 15
hours and 1.02 mL (982 mg, 9.52 mmol) of diethylenetriamine was
then added followed by 2.65 mL (1.93 g, 19.0 mmol) of Et.sub.3N.
The resulting mixture was stirred for 4 hours, and the solid
product was collected by filtration. Recrystallization (MeOH/EtOAc)
gave 4.27 g (75%) of compound 18 as a fine grainy solid: m.p.
132-133.degree.; .sup.1H NMR (CDCl.sub.3) .delta. 1.33 (m, 4H),
1.52 (m, 4H), 1.64 (m, 4H), 2.18 (t, 4H), 2.73 (t, 4H), 3.16 (m,
4H), 3.35 (m, 4H), 4.96 (bd s, 2H), 5.09 (s, 4H), 6.13 (bd s, 2H),
7.33 (s, 10H); Anal. calc'd for C.sub.32H.sub.47N.sub.5O.sub.6: C,
64.29; H, 7.50; N, 11.72. Found: C, 63.54; H, 7.75; N, 11.91.
[0148] Compound 19: 657 uL (880 mg, 3.2 mmol) of
triethyleneglycol-bis-chloroformate (Aldrich) was added to a
solution of 4.86 g (8.1 mmol) of compound 18 in 162 mL of pyridine
in a 20.degree. water bath. The mixture immediately formed a
precipitate. The mixture was stirred for 16 hours and the resulting
cloudy yellow solution was concentrated under vacuum. The
concentrate was partitioned between 150 mL of EtOAc and two 150 mL
portions of 1 N HCl (making sure the aqueous layer was acidic). The
aqueous layers were combined and extracted with a second 1.50 mL
portion of EtOAc. The EtOAc layers were combined, dried
(MgSO.sub.4), filtered, and concentrated. The resulting residue was
crystallized (EtOAc/hexanes/CHCl.sub.3) to provide 1.92 g (43%) of
compound 19 as fine yellow tinted crystals: m.p. 86-91.degree.;
.sup.1H NMR (CDCl.sub.3) 1.31 (m, 8H), 1.52 (m, 8H), 1.62 (m, 8H),
2.20 (m, 8H), 3.20 (m, 8H), 3.39 (s, 16H), 3.62 (s, 4H), 3.68 (m,
4H), 4.26 (m, 4H), 5.08 (s, 8H), 5.32 (bd s, 4H), 7.31 (bd s, 4H),
7.37 (s, 20H); .sup.13C NMR (CDCl.sub.3) .delta. 25.1, 26.2, 26.4,
29.6, 36.0, 36.2, 38.5, 38.8, 40.8, 64.5, 66.4, 69.1, 70.3, 128.0,
128.4, 136.7, 156.5, 156.9, 173.6; Anal. calc'd for
C.sub.72H.sub.104N.sub.10O.sub.18: C, 61.87; H, 7.50; N, 10.02.
Found: C, 61.68; H, 7.63; N, 9.95.
[0149] Compound 36: 3.5 mL of cyclohexene was added to a solution
of 800 mg (0.57 mmol) of compound 19 in 5 mL of absolute EtOH. The
solution was placed under nitrogen, 500 mg of 10% Pd on carbon was
added, and the resulting mixture was refluxed with stirring for 2
hours. When cool, the mixture was filtered through diatomaceous
earth and concentrated to give 500 mg (100%) of compound 36 as an
oil: .sup.1H NMR (50/50 CDCl.sub.3/CD.sub.3OD) .delta. 1.21 (m,
8H), 1.49 (m, 8H), 1.62 (m, 8H), 2.19 (t, J=7.4 Hz, 8H), 2.67 (t,
J=7.4 Hz, 8H), 3.36 (bd s, 16H), 3.67 (s, 4H), 3.71 (m, 4H), 4.21
(m, 4H).
[0150] Compound 20: 3.9 g (46.4 mmol) of NaHCO.sub.3 was added to a
solution of 5.0 g (5.8 mmol) of compound 36 in 37.5 mL of dioxane
and 12.5 mL of H.sub.2O. The mixture was cooled to 0.degree. in an
ice bath and 8.7 g (34.8 mmol) of 4-nitrophenylbromoacetate,
compound 10, was added. The mixture was stirred at 0.degree. for 1
hour and 50 mL of 1 N H.sub.2SO.sub.4 was slowly added. The mixture
was extracted with three, 50 mL portions of EtOAc. The EtOAc
extracts were discarded and the aqueous layer was extracted with
six, 50 mL portions of 20/80 MeOH/CH.sub.2Cl.sub.2. The combined
MeOH/CH.sub.2Cl.sub.2 layers were dried (Na.sub.2SO.sub.4),
filtered, and concentrated. The residue was purified by silica gel
chromatography (step gradient 9/1 CH.sub.2Cl.sub.2/MeOH then
85/15/5 CH.sub.2Cl.sub.2/MeOH/THF) to provide 3.62 g (46%) of
compound 20 as a white solid: melting point 66.0-70.5.degree.. An
analytical sample was prepared by preparative HPLC (C.sub.18
reversed phase column, gradient 25/75/0.1 to 35/65/0.1
CH.sub.3CN/H.sub.2O/CF.sub.3CO.sub.2H over 50 minutes, 225 .mu.m)
to give a clear oil which solidified on standing under vacuum to
give a white solid: melting point 87-89.degree.; .sup.1H NMR
(CDCl.sub.3) .delta. 1.35 (m, 8H), 1.55 (m, 8H), 1.64 (m, 8H), 2.26
(m, 8H), 3.28 (m, 8H), 3.42 (bd s, 16H), 3.66 (s, 4H), 3.70 (m,
4H), 3.89 (s, 8H), 4.19 (m, 4H); .sup.13C NMR (CDCl.sub.3) .delta.
25.1, 26.2, 28.8, 29.0, 38.5, 39.1, 40.0, 47.8, 48.3, 64.7, 69.1,
70.3, 157.0, 166.3, 174.9; MS (FAB) m/e (relative intensity)
MH+[1341 (25), [343 (60), 1345 (70), 1347 (56), 1349 (21)], 705.6
(100); Anal. calc'd for C.sub.48H.sub.84N.sub.10O.sub.14Br.sub.4:
C, 42.86; H, 6.29; N, 9.27; Br, 23.77. Found: C, 42.15; H, 6.28; N,
9.87; Br, 25.33.
[0151] Compound 21--[Tetrakis-(2-cyanoethoxymethyl)methane]: This
compound was prepared similarly to the method reported (Bruson, H.
A., U.S. Pat. No. 2,401,607; Jun. 4, 1946). 27.3 mL (21.8 g, 411
mmol) of acrylonitrile was added to a stirred solution of 8.0 g
(58.8 mmol) of pentaerythritol and 1.76 mL of a 40% aqueous
solution of benzyltrimethylammonium hydroxide in 50 mL of H.sub.2O.
A reflux condenser was affixed and the mixture was heated under
N.sub.2 atmosphere with stirring at 40.degree. for 16 hours and
then at 60.degree. for 24 hours. When cool, the mixture was
acidified with 1 mL of concentrated HCl and transferred to a
separatory funnel. The oil which settled to the bottom was
collected, and the aqueous phase was extracted with three 40 mL
portions of CH.sub.2Cl.sub.2. The oil and combined extracts were
dried (MgSO.sub.4), filtered, and concentrated to give 23.5 g of
oil. Biscyanoethyl ether was removed by Khugelrhor distillation at
110.degree. and 0.25 torr. The pot residue was crystallized from 1
L of H.sub.2O to give 8.43 g (41%) of compound 21 as white needles:
m.p. 42.5.degree. [Reported (Macromolecules 1991, 24, 1443-1444.)
39-40.degree.]; .sup.1H NMR (CDCl.sub.3) .delta. 2.61 (t, J=6 Hz,
8H), 3.50 (s, 8H), 3.6 (t, J=6 Hz, 8H).
[0152] Compound 22--[Tetrakis-(2-carboxyethoxymethyl)methane]: A
solution of 5.0 g (14.35 mmol) of compound 21 in 21.5 mL of
concentrated HCl was stirred at 75.degree. for 3 h; during this
time a white precipitate formed. The aqueous HCl was removed under
vacuum, and the mixture was concentrated twice from 25 mL of
H.sub.2O. The resulting 9.68 g of solid material was loaded onto a
45 mm i.d. column containing a 16.5 cm bed of DOW-1-X2 resin in the
hydroxide form, and the column was eluted with 200 mL of H.sub.2O
followed by 1 N HCl. Fractions containing product, as evidenced by
TLC (80/20/1 CH.sub.3CN/H.sub.2O/HOAc), were concentrated to give
1.21 g (21%) of 22 as an oil: .sup.1H NMR (D.sub.2O) .delta. 2.46
(t, J=6 Hz, 8H), 3.22 (s, 8H), 3.55 (t, J=6 Hz, 8H).
[0153] Compound 23: 3.71 mL (6.06 g, 50.8 mmol) of thionyl chloride
was added to a solution of 1.12 g (2.85 mmol) of compound 22 in 7.0
mL of THF. The mixture was stirred at room temperature for 3 hours
and the solvents were removed under vacuum. The crude acid chloride
was dissolved in 7 mL of THF. 2.12 mL (1.54 g, 15.24 mmol) of
Et.sub.3N was then added to the solution. The mixture was stirred
under N.sub.2 and cooled to 0.degree.. A solution of 3.60 g (12.74
mmol) of compound 4 in 5 mL of THF was added over a 1 minute
period. The cooling bath was removed, and the mixture was stirred
for 5.5 hours at room temperature and then partitioned between 25
mL of 1 N HCl and four 25 mL portions of EtOAc. The EtOAc layers
were combined, washed with brine, dried (MgSO.sub.4), filtered, and
concentrated to provide 3.46 g of viscous oil. Purification by
chromatography on silica gel (95/5 CH.sub.2Cl.sub.2/MeOH) provided
1.26 g (30%) of compound 23 as a viscous oil: .sup.1H NMR
(CDCl.sub.3) .delta. 2.40 (t, 8H), 3.29 (s, 8H), 3.35 (m, 16H),
3.48-3.77 (m, 48H), 5.12 (s, 8H), 5.60 (bd, 4H), 6.85 (bd, 4H),
7.34 (s, 20H).
[0154] Compound 37: 4.0 mL of cyclohexene and 83 mg of 10% Pd on
carbon were added to a solution of 142 mg (0.093 mmol) of compound
23 in 8.4 mL of EtOH under N.sub.2. The mixture was refluxed with
stirring in a 90.degree. oil bath for 3 hours and, when cool,
filtered through diatomaceous earth using CH.sub.2Cl.sub.2 to wash
the filter and flask. The filtrate was concentrated to provide 70
mg (78%) of compound 37 as an oil: .sup.1H NMR (CDCl.sub.3) .delta.
2.90 (t, 8H), 3.33 (s, 8H), 3.45 (t, 8H), 3.52-3.73 (m, 48H).
[0155] Compound 24: 40 mg (0.48 mmol) of NaHCO.sub.3 and 104 mg
(0.40 mmol) of compound 10 were added to a solution of 70 mg (0.098
mmol) of compound 37 in 2 mL of dioxane and 0.67 mL of H.sub.2O.
The mixture was stirred for 17 hours at room temperature and 0.5 mL
of 1 NH.sub.2SO.sub.4 was added, bringing the pH to 4. The mixture
was concentrated, and the concentrate was purified by
chromatography on G-10 Sephadex.RTM. (MeOH). The fractions
containing product were concentrated under vacuum to provide 91 mg
of oil. Purification of 36 mg of the crude product by HPLC
(C.sub.18, gradient 20/80/0.1 to 35/65/0.1
CH.sub.3CN/H.sub.2O/CF.sub.3CO.sub.2H) gave 19 mg (44%) of compound
24 as an oil: .sup.1H NMR (CDCl.sub.3) .delta. 2.50 (t, 8H), 3.31
(s, 8H), 3.36-3.72 (m, 56H), 3.91 (s, 8H); .sup.13C NMR
(CDCl.sub.3) .delta. 28.8, 36.5, 39.7, 40.0, 67.2, 69.3, 69.5,
70.3, 166.6, 173.0. MS (FAB) m/e (relative intensity) MH.sup.+[
1425(15), 1427(63), 1429(75), 1431(64), 1433(12)], 577(100).
[0156] Compound 25a--[Bis-tolsylate of PEG.sub.3350]: 6.47 mL of
pyridine was added to a solution of 16.75 g (5.0 mmol) of
polyethylene glycol (J. T. Baker, average molecular weight 3350 g
per mol) which had been dried by azeotropic distillation (toluene)
in 40 mL of CH.sub.2Cl.sub.2. The solution was placed under
nitrogen and cooled to 0.degree.. A solution of 7.63 g (40 mmol) of
tosyl chloride in 40 mL of CH.sub.2Cl.sub.2 was added over a 25
minute period. The cooling bath was removed and the mixture was
stirred at room temperature for 16 hours. The mixture was shaken
with 80 mL of 1 N HCl and the CH.sub.2Cl.sub.2 layer which
contained emulsions was washed with 100 mL of H.sub.2O. The
CH.sub.2Cl.sub.2 layer was dried (MgSO.sub.4), filtered, and
concentrated. The residue was crystallized from
CH.sub.2Cl.sub.2/Et.sub.2O to provide 16.82 g (92%) of compound 25a
as a white solid: .sup.1H NMR (CDCl.sub.3) d 2.50 (s, 6H), 3.48 (t,
J=5 Hz, 4H), 3.55-3.77 (m, more than 600H, integral too large to be
accurate), 3.83 (t, J=5 Hz, 4H), 7.44 (d, J=7 Hz, 4H), 7.94 (d, J=7
Hz, 4H).
[0157] Compound 26a--Diazido-PEG.sub.350: A solution of 10.83 g
(2.96 mmol) of compound 25a and 1.92 g (29.6 mmol) of NaN.sub.3 in
30 mL of DMF was heated under N.sub.2 in a 120.degree. oil bath for
3 hours. When cool, the mixture was partitioned between 100 mL of
H.sub.2O and 100 mL of CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layer
was diluted to 200 mL with CH.sub.2Cl.sub.2 and washed with 100 mL
of 1 N HCl, dried (Na.sub.2SO.sub.4), filtered and concentrated.
The resulting waxy solid was recrystallized
(CH.sub.2Cl.sub.2/Et.sub.2O), and the resulting solids were further
purified by chromatography on silica gel (gradient 98/2 to 95/5
CH.sub.2Cl.sub.2/MeOH) to provide 4.75 g (47%) of compound 26a as a
waxy solid: TLC Rf 0.41 (9/1 CH.sub.2Cl.sub.2); .sup.1H NMR
(CDCl.sub.3) .delta. 3.35 (t, J=5 Hz, 4H), 3.44 (t, J=5 Hz, 2H),
3.54-3.77 (m, approx. 300H, integral too large to be accurate),
3.79 (t, J=5 Hz, 2H).
[0158] Compound 27a--[Diamino-PEG.sub.3350]: 473 mg of 10% Pd on
carbon (Aldrich) was added to a solution of 4.75 g (1.39 mmol) of
compound 26a in 140 mL of EtOH. The mixture was shaken under 60 psi
of H.sub.2 for 30 hours. Because the reaction was incomplete (TLC,
9/1 CH.sub.2Cl.sub.2/MeOH), another 473 mg of 10% Pd on carbon was
added and the mixture was shaken under 60 psi of H.sub.2 for
another 5 hours. The mixture was then filtered through diatomaceous
earth, concentrated under vacuum, and the concentrate was
crystallized (CH.sub.2Cl.sub.2/Et.sub.2O) to give 4.03 g (86%) of
compound 27a as a white solid: .sup.1H NMR (CDCl.sub.3) .delta.
2.92 (t, 4H), 3.49 (t, 2H), 3.66 (t, 4H), 3.67 (m, approx. 300H,
integral too large to be accurate), 3.86 (t, 2H).
[0159] Compound 28--[N-hydroxysuccinimidyl ester of compound 7]:
596 mg (2.89 mmol) of dicyclohexylcarbodiimide was added to a
solution of 1.84 g (2.41 mmol) of compound 7 and 278 mg (2.41 mmol)
of NHS in 12 mL of THF at 0.degree. under N.sub.2. The cooling bath
was removed, and the mixture was stirred at room temperature for 16
hours. 250 uL of acetic acid was added to the mixture. Stirring was
continued at room temperature for 1 hour. The mixture was then
placed in a freezer for 2 hours. The solids were removed by
filtration, and the filtrate was concentrated to give 2.27 g (110%)
of crude compound 28 as a viscous oil. Compound 28 was difficult to
purify without decomposition, so it was used directly to acylate
diamino-PEG.
[0160] Compound 29a: A solution of 900 mg (1.05 mmol) of compound
28 in 4.68 mL of dioxane was added to a solution of 877 mg (0.26
mmol) of compound 27a and 176 mg (2.10 mmol) of NaHCO.sub.3 in 3.12
mL of H.sub.2O at 0.degree.. The mixture was stirred for 2 hours
and then partitioned between 25 mL of 1 N HCl and two 25 mL
portions of CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers
were dried (Na.sub.2SO.sub.4), filtered, and concentrated to give a
viscous oil. Purification by silica gel chromatography (gradient,
95/5 to 87/13 CH.sub.2Cl.sub.2/MeOH) yielded 695 mg (55%) of
compound 29a as a waxy solid: .sup.1H NMR (CDCl.sub.3) .delta. 2.55
(bd, 8H), 3.39 (m, 16H), 3.44-3.72 (m, approx. 432H, integral too
large to be accurate), 3.89 (s, 4H), 4.03 (s, 4H), 5.09 (s, 8H),
7.36 (s, 20H).
[0161] Compound 38a: 7.1 mL of cyclohexene was added to a solution
of 688 mg (0.142 mmol) of compound 29a in 14.2 mL of EtOH under
N.sub.2. 284 mg of 10% Pd on carbon was added and the resulting
mixture was refluxed for 2 hours. When cool, the mixture was
filtered through diatomaceous earth with EtOH, and the filtrate was
concentrated under vacuum to yield 550 mg (90%) of compound 38a as
a waxy solid: .sup.1H NMR (CDCl.sub.3) .delta. 2.58 (m, 8H), 2.93
(m, 8H), 3.38-376 (m, approx. 550H), 4.00 (s, 4H), 4.13, (s,
4H).
[0162] Compound 30a: A solution of 268 mg (1.04 mmol) of compound
10 in 4.65 mL of dioxane was added to a solution of 550 mg (0.13
mmol) of compound 38a and 175 mg (2.08 mmol) of NaHCO.sub.3 in 3.11
mL of H.sub.2O at 0.degree.. The mixture was stirred for 20 hours
and partitioned between 50 mL of 1 N H.sub.2SO.sub.4 and two 50 mL
portions of CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers
were dried (Na.sub.2SO.sub.4), filtered, and concentrated to an
oil. Purification by G-10 Sephadex.RTM. chromatography (MeOH) gave
an amorphous solid which was crystallized (EtOH/Et.sub.2O) to
provide 378 mg (61%) of compound 30a as a white solid: .sup.1H NMR
(CDCl.sub.3) .delta. 2.59 (bd s, 8H), 3.38-3.82 (m, approx. 500H,
integral too large to be accurate), 3.88 (s, 8H), 3.98 (s, 4H),
4.10 (s, 4H); bromoacetyl determination (European Journal of
Biochemistry, 1984, 140, 63-71): Calculated, 0.84 mmol/g; Found,
0.50 mmol/g.
[0163] Compound 25b--[Bis-tosylate of PEG.sub.8000]: 2.3 mL (16.5
mmol) of triethylamine, followed by 3.15 g (16.5 mmol) of TsCl, was
added to a solution of 12.0 g (1.5 mmol) of PEG.sub.8000 (Aldrich,
average molecular weight 8000 g/mmol) which had been dried by
azeotropic distillation (toluene) in 30 mL of CH.sub.2Cl.sub.2. The
mixture was stirred at room temperature for 18 hours and extracted
with four, 50 mL portions of 1 N HCl followed by 50 mL of saturated
NaCl solution. The CH.sub.2Cl.sub.2 layer was dried
(Na.sub.2SO.sub.4), filtered, and concentrated under vacuum to
provide a waxy solid. Recrystallization
(CH.sub.2Cl.sub.2/Et.sub.2O) gave 11.0 g (92%) of compound 25b as a
white solid: .sup.1H NMR (CDCl.sub.3) .delta. 2.38 (s, 6H),
3.40-3.89 (m. approx. 800H, integral too large to be accurate),
4.14 (m, 4H), 7.34 (d, J=8.2 Hz, 4H), 7.79 (d, J=8.2 Hz, 4H).
[0164] Compound 26b--[Diazido-PEG.sub.8000]: 1.86 g (28.6 mmol) of
NaN.sub.3 was added to a solution of 10.8 g (1.3 mmol) of compound
25b in 30 mL of dry DMF. The mixture was heated under N.sub.2 at
120.degree. for 2.5 hours. When cool, the mixture was partitioned
between 240 mL of CH.sub.2Cl.sub.2 and three 50 mL portions of 0.5
N HCl. The CH.sub.2Cl.sub.2 layer was washed with 50 mL of
saturated NaCl solution, dried (Na.sub.2SO.sub.4), filtered, and
concentrated to give a solid. Purification by chromatography on
silica gel (gradient 2/98 to 6/94 MeOH/CH.sub.2Cl.sub.2) and
recrystallization of the purified product (MeOH/Et.sub.2O) gave
6.95 (66%) of compound 26a as a white solid: TLC (Rf=0.33, 12/88
MeOH/CH.sub.2Cl.sub.2); .sup.1H NMR (CDCl.sub.3) 3.39-3.86 (m).
[0165] Compound 27b--[Diamino-PEG.sub.8000]: A solution of 6.9 g
(0.86 mmol) of compound 26b in 150 mL of MeOH saturated with
ammonia was sparged with nitrogen. 1.5 g of 10% Pd/C was added and
the mixture was shaken under 65 psi of H.sub.2. After 20 hours, TLC
analysis indicated that the reaction was incomplete. As a result,
200 mg of 10% Pd/C was added and shaking under 65 psi of H.sub.2
was continued for another 20 hours. The mixture was filtered
through diatomaceous earth and the filtrate was concentrated under
vacuum. The resulting waxy solid was recrystallized
(MeOH/Et.sub.2O) to give 6.0 g (89%) of compound 27b as a white
solid. .sup.1H NMR (CDCl.sub.3) .delta. 2.96 (t, J=5.1 Hz, 4H),
3.40-3.89 (m, approx. 700H, integral too large to be accurate).
[0166] Compound 29b: 221 mg (2.63 mmol) of NaHCO.sub.3 was added to
a solution of 3.0 g (0.375 mmol) of compound 27b in 10 mL of water
and 3 mL of dioxane. 1.3 g (1.51 mmol) of compound 28 dissolved in
10 mL of dioxane was then added. The mixture was stirred for 24
hours and then 40 mL of 0.5 N HCl was added. The mixture was
extracted with four, 25 mL portions of CH.sub.2Cl.sub.2. The
combined CH.sub.2Cl.sub.2 layers were dried (MgSO.sub.4), filtered,
and concentrated to an oil. Crystallization from MeOH/Et.sub.2O
provided 2.0 g (58%) of compound 29b: .sup.1H NMR (CDCl.sub.3) d
2.52 (m, 8H), 3.40-3.64 (m, approx. 700H, integral too large to be
accurate), 3.89 (s, 4H), 4.02 (s, 4H), 5.09 (s, 8H), 7.35 (s,
20H).
[0167] Compound 38b: 123 mg of 10% Pd/C was added to a solution of
600 mg (0.063 mmol) of compound 29b in 5 mL of absolute EtOH and
2.5 mL of cyclohexene under nitrogen. This mixture was refluxed
under nitrogen for 2 hours. The reaction mixture was filtered
through diatomaceous earth and evaporated to give 549 mg (97%) of
compound 38b as a white solid: .sup.1H NMR (CDCl.sub.3) .delta.
2.58 (m, 8H), 2.90 (m, 8H), 3.39-3.70 (m, approx. 700H, integral
too large to be accurate), 4.05 (s, 4H), 4.15 (s, 4H).
[0168] Compound 30b: 100 mg (1.2 mmol) of NaHCO.sub.3, followed by
84 mg (0.32 mmol) of compound 10, was added to a solution of 529 mg
(0.059 mmol) of compound 38b in 2 mL of dioxane and 5 mL of water.
After stirring for 12 hours, the reaction was acidified with 1 N
H.sub.2SO.sub.4 and extracted with four, 40 mL of CHCl.sub.3. The
combined CHCl.sub.3 layers were dried (MgSO.sub.4), filtered, and
concentrated to give 503 mg of semi-solid residue. The residue was
purified by chromatography on G-10 Sephadex.RTM. (MeOH) and
crystallized (MeOH/Et.sub.2O/hexanes) to give 215 mg (39%) of
compound 30b as a white solid: .sup.1H NMR (CDCl.sub.3) .delta.
2.58 (m, 8H, 3.35-3.70 (m, approx. 700H, integral too large to be
accurate), 3.89 (s, 8H), 4.01 (s, 4H), 4.16 (s, 4H); bromoacetyl
determination (European Journal of Biochemistry 1984, 140, 63-71):
Calculated, 0.42 mmol/g; Found, 0.27 mmol/g.
[0169] Compound 31--[PEG.sub.3350-bis-chloroformate]: Two drops of
dry pyridine, followed by 125 mg (0.418 mmol) of triphosgene, was
added to a solution of 1.0 gram (0.249 mmol) of polyethylene glycol
(J. T. Baker, average molecular weight 3350 g per mol) which had
been dried by azeotropic distillation (toluene) in 12 mL of
CH.sub.2Cl.sub.2. The mixture was stirred at room temperature for
20 hours and the solvent was evaporated under vacuum to give 1.0 g
(100%) of compound 31 as a white solid: .sup.1H NMR (CDCl.sub.3)
.delta. 3.40-3.65 (m, approx. 300H, integral too large to be
accurate), 3.77 (m, 4H), 4.46 (m, 4H).
[0170] Compound 32: A solution of 1.0 g (0.25 mmol) of compound 31
in 12 mL of 5:1 CH.sub.2Cl.sub.2/dioxane was added dropwise to a
50.degree. solution of 600 mg (1.0 mmol) of compound 18 in 10 mL of
dioxane and 1.5 mL of pyridine. The resulting cloudy solution was
stirred for 72 hours. 25 mL of CH.sub.2Cl.sub.2 was added and the
mixture was then filtered. The filtrate was evaporated and the
semi-solid residue was purified by chromatography on G-10
Sephadex.RTM.. The resulting solid was crystallized
(CH.sub.2Cl.sub.2/Et.sub.2O) to give 829 mg (75%) of compound 32 as
a faintly yellow solid: .sup.1H NMR (CDCl.sub.3) .delta. 1.30 (m,
8H), 1.40 (m, 8H), 1.61 (m, 8H), 2.18 (m, 8H), 3.17 (m, 8H), 3.40
(m, 16H), 3.62 (m, approx. 300H, integral too large to be
accurate), 4.15 (m, 4H), 5.07 (s, 8H), 7.33 (m, 20H).
[0171] Compound 39: 100 mg of 10% Pd/C was added to a solution of
300 mg (0.065 mmol) of compound 32 in 5 mL of absolute EtOH and 2
mL of cyclohexene under nitrogen. This mixture was refluxed under
nitrogen for 2 hours. The mixture was filtered through diatomaceous
earth and the solvent was evaporated to give 237 mg (90%) of
compound 39 as a white solid: .sup.1H NMR (CDCl.sub.3) .delta. 1.37
(m, 8H), 1.48 (m, 8H), 1.65 (m, 8H), 2.21 (m, 8H), 2.50 (m, 8H),
3.39 (m, 16H), 3.64 (m, approx. 300H, integral too large to be
accurate), 4.19 (m, 4H).
[0172] Compound 33: 125 mg (0.67 mmol) of NaHCO.sub.3 and 115 mg
(0.44 mmol) of compound 10 was added to a solution of 225 mg (0.055
mmol) of 39 in 5 mL of dioxane and 5 mL of water. The resulting
yellow solution was stirred at room temperature for 12 hours. The
solution was then extracted with three 30 mL portions of
CH.sub.2Cl.sub.2. The aqueous layer was acidified with 1 N
H.sub.2SO.sub.4 and extracted with three, 30 mL portions of
CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers were dried
(MgSO.sub.4), filtered, and concentrated to provide a yellow oil.
Purification by chromatography on G-10 Sephadex.RTM. (MeOH) and
recrystallization of the resulting oil (EtOH/Et.sub.2O) provided
182 mg (73%) of compound 33 as a white-solid: .sup.1H NMR
(CDCl.sub.3) .delta. 1.35 (m, 8H), 1.55 (m, 8H), 1.65 (m, 8H), 2.22
(m, 8H), 3.28 (m, 8H), 3.42 (m, 16H), 3.50-364 (m, approx. 300H,
integral too large to be accurate), 3.87 (s, 8H), 4.18 (m, 4H);
bromoacetyl determination (European Journal of Biochemistry 1984,
140, 63-71): Calculated, 0.87 mmol/g; Found, 0.73 mmol/g. Anal
Calcd. for C.sub.191H.sub.375O.sub.87N.sub.10Br.sub.4: C, 50.84; H,
8.33; N, 3.09; Br, 7.05. Found: C, 51.98; H, 8.34; N, 2.45; Br,
10.19.
[0173] Compound 40--[4-Nitrophenyliodoacetate]: 5.15 g (25 mmol) of
dicyclohexylcarbodiimide and 2.92 g (2.92 mmol) of 4-nitrophenol in
100 mL of EtOAc were added to a 0.degree. solution of 3.72 g (20
mmol) of iodoacetic acid. The mixture was stirred at 0.degree. for
1 hour and at room temperature for 2 hours. The solids were removed
by filtration, and the filtrate was concentrated under vacuum. The
resulting yellow solid was recrystallized (EtOAc/hexanes/trace
HOAc) to yield 4.82 g (78%) of compound 40 as a yellow-brown solid:
.sup.1H NMR (CDCl.sub.3) .delta. 4.00 (s, 2H), 7.39 (d, 2H), 8.40
(m, 2H).
[0174] Compound 41: 103 mg (1.22 mmol) of NaHCO.sub.3, followed by
211 mg (0.692 mmol) of compound 40, was added to a solution of 110
mg (0.104 mmol) of compound 34 in 5 mL of dioxane and 5 mL of
H.sub.2O. The mixture was stirred for 18 hours and then
concentrated under vacuum. Purification by chromatography on
Sephadex.RTM. (MeOH) provided 140 mg (87%) of compound 41 as an
oil. An analytical sample was prepared by preparative HPLC
(C.sub.18, gradient 20/80/0.1 to 25/75/0.1 CH.sub.3CN/H.sub.2O/TFA
over 60 minutes, 225 nm): .sup.1H NMR (CDCl.sub.3) .delta. 2.59 (m,
4H), 2.65 (m, 4H), 3.44-3.62 (m, 60H), 3.77 (s, 4H), 3.78 (s, 4H),
4.02 (s, 4H), 4.21 (s, 4H).
[0175] Compound 42: 145 mg (0.935 mmol) of
N-methoxycarbonylmaleimide was added with vigorous stirring to a
solution of 171 mg (0.161 mmol) of compound 34 in 8 mL of dioxane,
2 mL of saturated NaHCO.sub.3 solution, and 2 mL of H.sub.2O at
0.degree. (The Practice of Peptide Synthesis, M. Bodansky and A.
Bodansky, Springer-Verlag, New York, 1984, pages 29-31. Keller, O.,
Rudinger, J. Helv. Chim. Acta 1975, 58, 531.). After 15 minutes, 25
mL of dioxane was added, the cooling bath was removed, and stirring
was continued for 45 minutes at room temperature. The mixture was
extracted with two, 30 mL portions of CHCl.sub.3 and the combined
CHCl.sub.3 layers were dried (MgSO.sub.4), filtered, and
concentrated to an oil. Purification by chromatography on G-10
Sephadex.RTM. (MeOH) gave 103 mg (45%) of compound 42 as an oil. An
analytical sample was prepared by preparative HPLC (C.sub.18,
gradient 20/80/0.1 to 25/75/0.1 CH.sub.3CN/H.sub.2O/TFA over 65
minutes, 225 nm) to give an oil: .sup.1H NMR (CDCl.sub.3) .delta.
2.57 (m, 4H), 2.67 (m, 4H), 3.42-3.65 (m, 52H), 3.72 (m, 8H), 4.03
(s, 4H), 4.17 (s, 4H), 6.74 (s, 4H), 6.75 (s, 4H).
[0176] Compound
43--[hydroxymethyl-tris-(2-cyanoethoxymethyl)methane]: 0.30 g (5.41
mmol) of KOH, followed by 23 mL (18.6 g, 350 mmol) of
acrylonitrile, was added to a solution of 6.8 g (50 mmol) of
pentaerythritol in 50 mL of H.sub.2O. The mixture was stirred at
room temperature for 16 hours, acidified with 1.5 mL of
concentrated HCl solution, and extracted with two, 50 mL portions
of CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers were
dried (MgSO.sub.4), filtered, and concentrated to give 16.97 g of
liquid. Purification by chromatography on silica gel (EtOAc)
yielded 8.49 g (51%) of compound 43 as a viscous oil: TLC, Rf=0.15
(EtOAc); .sup.1H NMR (CDCl.sub.3) .delta. 2.62 (t, 6H), 3.54 (s,
6H), 3.68 (t, 6H), 3.70 (s, 2H).
[0177] Compound
44--[hydroxymethyl-tris-(2-carboxymethylethoxymethyl)methane: 78 mL
of a saturated solution of HCl in MeOH was added to 5.45 g (15.6
mmol) of compound 43. The mixture was heated at reflux for 1 hour
and, when cool, partitioned between 100 mL of H.sub.2O and four,
100 mL portions of Et.sub.2O. The combined Et.sub.2O layers were
washed successively with 100 mL of saturated NaHCO.sub.3 solution
and 100 mL of saturated NaCl solution, dried (MgSO.sub.4),
filtered, and concentrated to yield 4.74 g of viscous liquid.
Purification by chromatography on silica gel provided 3.05 g (50%)
of compound 44 as an oil: TLC, Rf=0.27 (80/20 EtOAc/hexanes);
.sup.1H NMR (CDCl.sub.3) .delta. 2.58 (t, 6H), 3.43 (s, 6H), 3.61
(s, 2H), 3.69 (t, 6H), 3.70 (s, 9H); .sup.13C NMR (CDCl.sub.3)
.delta. 34.8, 44.9, 51.6, 65.2, 66.9, 71.0, 172.1.
[0178] Compound 45: A mixture of 560 mg (1.4 mmol) of compound 44
and 1.69 g (6.0 mmol) of compound 4 was heated under nitrogen at
150.degree. for 4 hours. The mixture was partitioned between 50 mL
of EtOAc and 25 mL of 1N HCl, and the HCl layer was extracted with
25 mL of CH.sub.2Cl.sub.2. Combined EtOAc and CH.sub.2Cl.sub.2
extracts were washed with saturated NaHCO.sub.3 solution, dried
(K.sub.2CO.sub.3), filtered, and concentrated to a viscous residue.
Purification by chromatography on silica gel (gradient 95/5 to
90/10 CH.sub.2Cl.sub.2/MeOH) provided 300 mg (19%) of compound 45
as a viscous oil: TLC, Rf=0.24 (90/10 CH.sub.2Cl.sub.2/MeOH);
.sup.1H NMR (CDCl.sub.3) .delta. 2.40 (t, 6H), 3.38 (s, 6H),
3.39-3.48 (m, 12H), 3.52-3.67 (m, 32H), 5.13 (s, 6H), 5.62 (bd s,
3H) 6.80 (bd s, 3H), 7.40 (s, 15H).
[0179] Compound 46: 104 mg of 10% Pd/C was added to a solution of
308 mg (0.269 mmol) of compound 45 in 10.4 mL of EtOH and 5.2 mL of
cyclohexene under nitrogen. A reflux condenser was attached and the
mixture was heated in an 85.degree. oil bath for 1.5 hours. When
cool, the mixture was filtered through diatomaceous earth and the
filtrate was concentrated to provide 177 mg of residue. The residue
was partially dissolved in 5.98 mL of dioxane. The resulting
mixture was added to 386 mg (1.49 mmol) of compound 10 followed by
a solution of 251 mg (2.99 mmol) of NaHCO.sub.3 in 3.99 mL of
H.sub.2O. The resulting mixture was stirred under nitrogen for 18
hours and partitioned between 25 mL of 1N HCl and three 25 mL
portions of CH.sub.2Cl.sub.2. The aqueous phase was extracted with
three 25 mL portions of 3/1 CH.sub.2Cl.sub.2/MeOH and three 25 mL
portions of 1/1 CH.sub.2Cl.sub.2/MeOH. The first two
CH.sub.2Cl.sub.2 extracts were discarded and the remaining extracts
were combined, dried (Na.sub.2SO.sub.4), filtered, and concentrated
to give 102 mg of a viscous oil. Purification by HPLC (C.sub.8,
23/77/0.1 CH.sub.3CN/H.sub.2O/CF.sub.3CO.sub.2H, 234 nm detection)
provided 43 mg (14%) of compound 46 as a viscous oil: .sup.1H NMR
(CDCl.sub.3) .delta. 2.48 (t, 6H), 3.40 (s, 6H), 3.44-3.54 (m,
14H), 3.56-3.62 (m, 12H), 3.63 (s, 12H), 3.67 (t, 6H), 3.91 (s,
6H), 6.90 (t, 3H), 7.10 (t, 3H); MS (FAB) m/e (relative intensity)
MH.sup.+[1103(17), 1105(42), 1107(41), 1109(18)],
MNa.sup.+[1125(38), 1127(100), 1129(99), 1131(39)].
[0180] Compound 47--S-(6-hydroxyhexyl)isothiuronium chloride: 11.1
g (146 mmol) of thiourea was added to a solution of 16.6 mL (20.0
g, 146 mmol) of 6-chlorohexanol in 49 mL of ethanol and the mixture
was refluxed for 24 hours. The mixture was cooled to 0.degree. and
the product crystallized. The crystals were collected by vacuum
filtration and dried to give 28.4 g (92%) of compound 47 as a white
solid: mp 122-124.degree.; .sup.1H NMR (DMSO) 1.40 (m, 4H), 1.65
(m, 2H), 3.21 (t, 2H), 3.41 (t, 2H), 9.27 and 9.33 (overlapping
broad singlets, 4H); Anal. Calc'd for C.sub.7H.sub.17ClN.sub.2OS:
C, 39.51; H, 8.06; N, 13.17; S, 15.07. Found: C, 39.69; H, 8.00; N,
13.01; S, 15.16.
[0181] Compound 48--6-Mercaptohexan-1-ol: 9.25 g of NaOH pellets
was added to a solution of 17.8 mg (83.6 mmol) of compound 47 in
120 mL of H.sub.2O and 120 mL of EtOH. The mixture was refluxed for
4 hours. The mixture was carefully concentrated to approximately 75
mL and the concentrate was purified by vacuum distillation to
provide 7.4 g (66%) of compound 48: bp 95-105.degree.@5 mm Hg;
.sup.1H NMR (CDCl.sub.3) 1.41 (m, 9H), 2.59 (dt, 2H), 3.69 (t with
underlying brd s, 3H); .sup.13C NMR (CDCl.sub.3) .delta. 24.5,
25.2, 28.0, 32.5, 33.9, 62.7; Anal. calc'd for C.sub.6H.sub.14OS:
C, 53.68, H, 10.51; S, 23.89. Found: C, 53.35; H, 10.72; S,
23.60.
[0182] Compound 49--Bis-(6-hydroxyhexyl)disulfide: A solution of
4.02 g (15.8 mmol) of 12 in 90 mL of MeOH was added dropwise over a
period of 10 minutes to a solution of 4.26 g (31.7 mmol) of
compound 48 in 10 mL of MeOH and 13.7 mL (9.97 g, 98.5 mmol) of
Et.sub.3N under N.sub.2 atmosphere and cooled in an ice bath. The
cooling bath was removed and the mixture was stirred at ambient
temperature for 4 hours. The mixture was concentrated on the rotary
evaporator and purified by silica gel chromatography (1:1
hexane/EtOAc) to provide 3.12 g (73%) of compound 49 as a pale
yellow solid: TLC R.sub.f0.18 (1:1 hexane/EtOAc); mp 38-48.degree.;
.sup.1H NMR (CDCl.sub.3) 1.15-2.20 (m, 16H), 2.73 (t, 4H), 3.70 (t,
4H); Anal. calc'd for C.sub.12H.sub.26S.sub.2O.sub.2: C, 54.09; H,
9.84; S, 24.06. Found: C, 54.85, H, 9.86; S, 24.11.
[0183] Compound
50--Mono-O-(4',4''-dimethoxytriphenylmethyl)-bis-(6-hydroxyhexyl)disulfid-
e: 3.97 g (11.7 mmol) of 4,4'-dimethoxytriphenylmethyl chloride was
added to a solution of 3.12 g (11.7 mmol) of compound 49 and 45 mL
of pyridine. The mixture was stirred at ambient temperature for 16
hours. Most of the pyridine was removed on the rotary evaporator
and the residue was partitioned between 100 mL of saturated
NaHCO.sub.3 solution and 100 mL of EtOAc. The EtOAc layer was
washed with 50 mL of saturated NaCl solution, dried
(Na.sub.2SO.sub.4), filtered and concentrated to an oil.
Purification by silica gel chromatography (9:1
CH.sub.2Cl.sub.2/EtOAc) yielded 2.84 g (43%) of compound 50 as a
viscous oil: TLC R.sub.f0.35 (9:1 CH.sub.2Cl.sub.2/EtOAc); .sup.1H
NMR (CDCl.sub.3) 1.41 (m, 8H), 1.65 (m, 8H), 2.70 (two overlapping
triplets, 4H), 3.08 (t, 2H), 3.65 (t, 2H), 3.81 (s, 6H), 6.85 (d,
4H), 7.32 (m, 7H), 7.47 (d, 2H); HRMS (FAB, M+), calc'd for
C.sub.33H.sub.44O.sub.4S.sub.2: 568.2681. Found: 568.2665.
Compound
51--O-[14-(4',4''-Dimethyoxytriphenylmethoxy)-7,8-dithiotetradec-
yl]-O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite: 458 mg (1.52
mmol) of O-cyanoethyl-N,N,N',N'-tetra-isopropylphosphorodiamidite
in 0.5 mL of CH.sub.2Cl.sub.2 was added to a solution of 771 mg
(1.36 mmol) of compound 50 and 116 mg (0.68 mmol) of
diisopropylammonium tetrazolide in 6.8 mL of CH.sub.2Cl.sub.2 under
N.sub.2 atmosphere. The mixture was stirred for 4 hours and
partitioned between 25 mL of NaHCO.sub.3 and 3.times.25 mL of
CH.sub.2Cl.sub.2. The combined CH.sub.2Cl.sub.2 layers were washed
with saturated NaCl solution, dried (Na.sub.2CO.sub.3), filtered
and concentrated to an oil. Purification by filtration through a
2'' plug of basic alumina in a 25 mm column, eluting with 9:1
CH.sub.2Cl.sub.2/Et.sub.3N provided 831 mg (80%) of compound 51 as
a viscous oil: .sup.1H NMR (CDCl.sub.3) .delta. 1.25 (m, 12H), 1.45
(m, 8H), 1.70 (m, 8H), 2.72 (m, 6H), 3.09 (t, 2H), 3.65 (m, 4H),
3.87 (s, 6H), 3.91 (m, 2H), 6.89 (d, 4H), 7.35 (m, 7H), 7.49 (d,
2H); .sup.31P NMR (CDCl.sub.3 with 15% H.sub.3PO.sub.4 internal
standard) 147.69; HRMS (FAB, MH+) calc'd for
C.sub.42H.sub.62N.sub.2O.sub.5PS.sub.2 769.3839, found
769.3853.
[0184] Compound 52--Trityl-HAD alcohol: 60 g (0.21 mol) of trityl
chloride was added to a solution of 57 g (0.21 mole) of compound 49
and 60 mL of pyridine. This mixture was stirred at 100.degree. C.
for 19 hours. The reaction mixture was cooled to room temperature
and filtered. The filtrate was diluted with 300 mL of methylene
chloride and extracted by 200 mL of saturated sodium bicarbonate.
The organic layer was dried over Na.sub.2SO.sub.4, filtered and
concentrated to an oil. Purification by silica gel chromatography
(gradient 9:1 hexanes:ethyl acetate 3:1 hexanes:ethyl acetate)
yielded 55 g of compound 52 (50%): .sup.1H NMR (CDCl.sub.3) .delta.
1.38 (m, 8H), 1.63 (m, 8H), 2.66 (m, 4H), 3.04 (t, 2H), 3.62 (t,
2H), 7.25 (m, 9H), 7.42 (m, 6H). HRMS (FAB, M+) calc'd for
C.sub.31H.sub.40O.sub.2S 508.2470, found 508.2482.
[0185] Compound 53--Trityl HAD Phosphoramidite: To a solution of 10
g (19.7 mmol) of compound 52 and 6.3 mL (36.2 mmol) of
diisopropylethylamine in 90 mL of methylene chloride at 0.degree.
C. under argon was slowly added 4.5 mL (20.2 mmol) of
2-cyanoethyl-N,N-diisopropylchlorophosphoramidite. After stirring
for 90 minutes, the reaction mixture was extracted twice with 100
mL of saturated sodium bicarbonate. The methylene chloride solution
was dried over Na.sub.2SO.sub.4, filtered and concentrated to an
oil. Purification by basic alumina:chromatography (75:24:1,
hexanes:ethyl acetate:triethylamine) provided 11.3 g (81%) of
compound 53 as an oil: .sup.1H NMR (CDCl.sub.3) .delta. 1.18 (m,
12H), 1.37 (m, 8H), 1.62 (m, 8H), 2.6 (m, 6H), 3.04 (t, 2H), 3.60
(m, 4H), 3.82 (m, 2H), 7.26 (m, 6H), 7.44 (m, 9H). HRMS (FAB, MH+)
calc'd for C.sub.40H.sub.58N.sub.2O.sub.3PS.sub.2 709.3626, found
709.3621.
[0186] Compound 54--O-(tert-butyldimethylsilyl)-5-hexenol: 15.66 g
(230 mmol) of imidazole and 20.0 g (130 mmol) of
tert-butyldimethylsilyl chloride were added to a solution of 12.47
mL (10.4 g, 104 mmol) of 5-hexene-1-ol in 104 mL of DMF. The
mixture was stirred at ambient temperature for 4 hours and
partitioned between 200 mL of EtOAc and 100 mL of saturated
NaHCO.sub.3 solution. The EtOAc layer was washed with 100 mL of
saturated NaHCO.sub.3 solution, 100 mL of saturated NaCl solution,
dried (MgSO.sub.4), filtered, and concentrated to a volume of
approximately 100 mL. Distillation under vacuum provided 70.07 g
(90%) of compound 54: bp 130-143.degree.@100 mm Hg; .sup.1H NMR
(CDCl.sub.3) 0.11 (s, 6H), 0.95 (s, 9H), 1.48 (m, 2H), 1.57 (m,
2H), 2.11 (dt, 2H), 3.66 (t, 2H), 5.03 (m, 2H), 5.86 (m, 1H);
.sup.13C NMR (CDCl.sub.3) --5.25, 18.40, 25.21, 26.01, 32.35,
33.60, 63.09, 114.40, 138.92; Anal. calc'd for C.sub.12H.sub.26OSi:
C, 67.22; H, 12.22. Found: C, 66.96; H, 12.16.
[0187] Compound
55--1-O-(tert-butyldimethylsilyl)-1,5,6-hexanetriol: To a solution
of 9.86 g (46.0 mmol) of compound 54 in 92 mL of acetone was added
a solution of 6.46 g (55.2 mmol) of N-methylmorphoholine oxide in
23 mL of H.sub.2O. To the mixture was added 443 .mu.l of a 2.5%
solution of OSO.sub.4 in tert-butyl alcohol (360 mg of solution,
9.0 mg of OSO.sub.4, 35 .mu.mol) and 50 .mu.L of 30%
H.sub.2O.sub.2. The mixture was stirred for 16 h and a solution of
474 mg of sodium dithionite in 14 mL of H.sub.2O was added. After
another 0.5 h the mixture was filtered through celite. The filtrate
was dried with MgSO.sub.4 and filtered through 1'' of silica gel in
a 150 mL Buchner funnel using 250 mL portions of EtOAc to elute.
Fractions containing product were concentrated to provide 11.0 g
(96%) of 55 as a viscous oil: TLC R.sub.f 0.2 (1:1 hexane/EtOAc);
.sup.1H NMR (CDCl.sub.3) 0.05 (s, 6H), 0.89 (s, 9H), 1.25 (m, 4H),
1.55 (m, 2H), 3.41 (dd, 2H), 3.62 (t, 2H), 3.71 (m, 1H); .sup.13C
NMR (CDCl.sub.3)-5.23, 18.42, 21.91, 26.02, 32.68, 32.81, 63.16,
66.74, 72.24; HRMS (FAB, MH+), calc'd for
C.sub.12H.sub.29O.sub.3Si: 249.1886. Found: 249.1889.
[0188] Compound
56--5,6-(bis-O-benzoyl)-1-O-(tert-butyldimethylsilyl)-1,5,6-hexanetriol:
6.18 mL (7.48 g, 53.2 mmol) of benzoyl chloride was added to a
solution of 5.29 g (21.3 mmol) of 55 in 106 mL of pyridine. The
mixture was stirred for 18 hours and concentrated on the rotary
evaporator. The mixture was partitioned between 100 mL of cold 1 N
HCl and 100 mL of EtOAc. The pH of the aqueous layer was checked to
make sure it was acidic. The EtOAc layer was washed successively
with 100 mL of H.sub.2O and 100 mL of saturated NaCl, dried
(MgSO.sub.4), filtered, and concentrated to provide 10.33 g (99%)
of compound 56 as a viscous yellow oil: TLC R.sub.f 0.45 (1:4
EtOAc/hexanes); .sup.1H NMR (CDCl.sub.3) .delta. 0.05 (s, 6H), 0.88
(s, 9H), 1.59 (m, 4H), 1.85 (m, 2H), 3.14 (t, 2H), 4.49 (dd, 1H),
4.59 (dd, 1H), 5.54 (m, 1H), 7.45 (m, 4H), 7.58 (m, 2H), 8.05 (m,
4H).
[0189] Compound 57--5,6-(bis-O-benzoyl)-1,5,6-hexanetriol: 10.7 mL
(10.7 mmol) of 1 N tetrabutylammonium fluoride in THF was added to
a solution of 2.62 g (5.36 mmol) of compound 56 in 10.9 mL of THF.
The mixture was stirred for 16 hours. The mixture was partitioned
between 25 mL of saturated NaHCO.sub.3 solution and 3.times.25 mL
of EtOAc. The combined EtOAc extracts were washed with saturated
NaCl solution, dried (MgSO.sub.4), filtered and concentrated to a
viscous oil which was purified by silica gel chromatography (1:1
hexane/EtOAc) to provide 823 mg (41%) of compound 57 as a viscous
oil; R.sub.f 0.14 (1:1 hexane/EtOAc); .sup.1H NMR (CDCl.sub.3)
.delta. 1.58 (m, 2H), 1.68 (m, 2H), 1.88 (m, 2H), 3.68 (t, 2H),
4.52 (dd, 1H), 4.62 (dd, 1H), 5.56 (m, 1H), 7.46 (m, 4H), 7.58 (m,
2H), 8.05 (m, 4H); .sup.13C NMR (CDCl.sub.3) .delta. 22.08, 31.20,
31.30, 32.88, 62.92, 66.17, 72.63, 128.93, 130.19, 130.57, 133.62,
166.72, 166.86; HRMS (FAB MH+), calc'd for C.sub.20H.sub.23O.sub.5;
343.1545. Found: 343.1553.
[0190] Compound
58--O-[5,6-(bis-O-benzoyloxy)-hexyl]-O-(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite: A solution of 989 mg (3.28 mmol) of
O-cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiamidite in 2.0 mL
of CH.sub.2Cl.sub.2 was added to a solution of 1.02 g (2.98 mmol)
of compound 57 and 255 mg (1.49 mmol) of diisopropylammonium
tetrazolide (prepared by mixing acetonitrile solutions of
diisopropylamine and tetrazole in a one-to-one mole ratio and
concentrating to a white solid) in 14.9 mL of CH.sub.2Cl.sub.2. The
mixture was stirred for 4 hours and then partitioned between 25 mL
of CH.sub.2Cl.sub.2 and 25 mL of chilled saturated NaHCO.sub.3
solution. The CH.sub.2Cl.sub.2 layer was washed with saturated NaCl
solution, dried (Na.sub.2SO.sub.4), filtered, and concentrated.
Purification by filtration through a 2'' plug of basic alumina in a
25 mm column, eluting with 9:1 EtOAc/Et.sub.3N, provided 1.5 g
(93%) of compound 58 as a viscous oil: .sup.1H NMR (CDCl.sub.3)
.delta. 1.19 (m, 12H), 1.62 (m, 2H), 1.73 (m, 2H), 1.90 (m, 2H),
2.62 (dd, 2H), 3.53-3.92 (m, 6H), 4.53 (dd, 1H), 4.62 (dd, 1H),
5.58 (m, 1H), 7.48 (m, 4H), 7.60 (m, 2H), 8.09 (m, 4H); .sup.31P
NMR (CDCl.sub.3 with 15% H.sub.3PO.sub.4 internal standard) d
148.2; HRMS (FAB, MH+), calc'd for C.sub.29H.sub.40O.sub.6N.sub.2P
543.2624. Found, 543.2619.
[0191] Compound 59--[4(iodoacetamido)benzoic acid: This compound
was prepared as described by Weltman, J. K., 1983 Biotechniques
1:148-152. Briefly, 708 mg (2.0 mmol) of iodoacetic anhydride was
added to a solution of 137 mg (1.0 mmol) of para-aminobenzoic acid
in 10 mL of dioxane. The mixture was stirred in the dark for 18
hours and partitioned between 25 mL of H.sub.2O and 25 mL of EtOAc.
The EtOAc layer was washed with saturated NaCl solution, dried
(MgSO.sub.4), filtered and concentrated to yield 797 mg of a peach
colored solid. Recrystallization from hexanes/EtOAc yielded 221 mg
(72%) of 4-(iodoacetamido)benzoic acid as a white solid: mp
220-230.degree.; .sup.1H NMR (CDCl.sub.3) .delta. 3.86 (s, 2H),
7.68 (d, 2H), 7.91 (d, 2H), 10.60 (s, 1H).
[0192] Compound 60--[4-(iodoacetamido)benzoyl derivative of
.alpha.,.omega.-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol:
188 mg (0.909 mmol) of dicyclohexylcarbodiimide was added to a
solution of 185 mg (0.606 mmol) of 4-(iodoacetamido)benzoic acid
and 406 mg (0.121 mmol) of
.alpha.,.omega.-bis-(N-2-aminoethylcarbamoyl)polyethyleneglycol
(Sigma Chemical Co., St. Louis, Mo., dried by azeotropic
distillation with toluene) in 2 mL of THF. The mixture was stirred
for 2 hours and then six drops of acetic acid were added. 10 mL of
CH.sub.2Cl.sub.2 was added and the mixture was kept in a freezer
for 30 minutes. The mixture was filtered to remove solids and the
filtrate was concentrated to a viscous residue. Purification by
silica gel chromatography (gradient 99/1 to 96/4
CH.sub.2Cl.sub.2/MeOH) provided a solid which was triturated with
MeOH to give 292 mg of a cream colored solid: .sup.1H (CDCl.sub.3)
3.48 (m, 8H), 3.63 (bd s, (CH.sub.2CH.sub.2O).sub.n, integral too
large to integrate), 3.98 (s, 4H), 4.18 (bd m, 4H), 5.91 (bd m,
2H), 7.48 (bd m, 2H), 7.76 (d, 4H), 7.88 (d, 4H), 9.38 (bd m, 2H):
iodoacetyl determination (European Journal of Biochemistry 1984,
140, 63-71): Calculated, 0.46 mmol/g; Found, 0.37 mmol/g.
EXAMPLE 3
Preparation of Activated Valency Platform Molecules and
Conjugates
[0193] There are many ways to form conjugates of biological or
chemical molecules and valency platform molecules. A particularly
specific method uses a thiol attached to the biological or chemical
molecule to react nucleophilically with a reactive "thiophillic"
group on the valency platform molecule to form a thioether bond,
but other combinations of reactive groups on the platform molecule
and on the biological or chemical molecule can also be employed for
attaching biological or chemical molecules covalently to a valency
platform molecule. Table 1 contains a number of combinations of
mutually reactive groups. The preference of any given method is
dictated by the nature of the biological or chemical molecule
(solubility, presence of other reactive groups, etc.).
TABLE-US-00001 TABLE 1 Nucleophile Mutually Reactive Group amine,
hydrazide active ester, anhydride, acid halide, hydrazine sulfonyl
halide, imidate ester, isocyanate, isothiocyanate, chloroformate
carbodiimide adduct, aldehyde, ketone sulfhydryl haloacetyl, alkyl
halide, alkyl sulfonate, maleimide, .alpha.,.beta.- unsaturated
carbonyl, alkyl mercurial, sulfhydryl, .alpha.,.beta.- unsaturated
sulfone
[0194] The following examples illustrate how various valency
platform molecules can be synthesized and conjugated with
biological or chemical molecules. These examples show how peptides
and oligonucleotides can be conjugated to valency platform
molecules using some of the mutually reactive groups in Table 1. In
addition to peptides and oligonucleotides, other biologically
active molecules (proteins, drugs, etc.) can also be conjugated to
valency platform molecules.
Combination 1: Thiol on Platform--Thiophile on Ligand
##STR00015##
[0196] Compound A: Compound 36 (861 mg, 1.0 mmol) and 252 mg (3.0
mmol) of NaHCO.sub.3 are dissolved in 20 mL of 1/1
dioxane/H.sub.2O. The mixture is cooled to 0.degree., and a
solution of 1.16 g (5.0 mmol) of N-succinimidyl-5-acetylthioacetate
(Prochem Inc.) in 40 mL of dioxane is added to the stirred mixture.
After 1 hour the mixture is extracted with CH.sub.2Cl.sub.2. The
combined extracts are dried (MgSO.sub.4), filtered, and
concentrated. The crude product is purified by silica gel
chromatography to provide A.
[0197] Compound B--Platform with Four Thiol Groups. A solution of
732 mg (0.55 mmol) of A in 7.3 mL of DMSO is added to 55 mL of
helium sparged pH 10, 100 mM sodium carbonate, 10 mM NH.sub.2OH
buffer. The mixture is kept under N.sub.2 and stirred for 1 hour to
obtain an approximately 10 mM solution of tetra-thiol platform
B.
[0198] Compound X--Bromoacetylated Peptide: A peptide is
synthesized with standard solid phase methods on a Wang
(p-alkoxybenzyl) resin using FMOC chemistry. FMOC protected amino
acids are added sequentially to the amino terminus. The final step
involves coupling N-bromoacetylaminocaproic acid. The protecting
groups are removed, and the peptide is removed from the resin with
trifluoroacetic acid to give X which is purified by preparative
reverse phase HPLC.
[0199] Peptide--Platform Conjugate, C. To the approximately 10 mmol
solution of tetrathiol platform, B, in pH 10 buffer, is added an
excess of a solution of bromoacetylated peptide, X, in DMSO. The
peptide conjugate, C, is purified by preparative reverse phase
HPLC.
Combination 2: Amine on Platform--Activated Carboxylate on
Peptide
##STR00016##
[0201] Compound Y--Peptide with Activated Carboxylate. A peptide is
synthesized with standard solid phase methods on a Wang
(p-alkoxybenzyl) resin, using TFA stable protecting groups (benzyl
ester on carboxyl groups and CBZ on amino groups). Amino acid
residues are added sequentially to the amino terminus. The peptide
is removed from the resin with TFA to provide a peptide with one
free carboxyl group at the carboxy terminus and all the other
carboxyls and amines blocked. The protected peptide, Y, is purified
by reverse phase HPLC.
[0202] Peptide--Platform Conjugate D. Compound Y (0.3 mmol) is
dissolved in 1 mL of DMF, and to the solution is added 0.3 mmol of
diisopropylcarbodiimide and 0.3 mmol of HOBT. The solution is added
to a solution of 0.025 mmol tetraamino platform, 36, in 1 mL of
DMF. When complete, the DMF is removed under vacuum to yield a
crude fully protected conjugate. The conjugate is dissolved in
MeOH, and the solution is placed in a Parr hydrogenation apparatus
with 100 mg of 10% Pd/C per gram of conjugate. The mixture is
shaken under 60 psi H.sub.2, and the deprotected conjugate, D, is
purified by preparative reverse phase HPLC.
Combination 3: Amine on Platform--Aldehyde on Oligonucleotide
##STR00017##
[0204] Oligonucleotide--Platform Conjugate, E. A 500 uL aliquot
(100 umol) of a 200 mM solution of NalO.sub.4 is added to a
solution of 1.0 g (400 mg of full length, 25 umol) of ACT-modified
(CA).sub.25 in 19.5 mL of H.sub.2O at 0.degree. in the dark. The
mixture is kept at 0.degree. for 40 minutes, and 50 mL of EtOH is
added. The mixture is kept at -20.degree. for 30 minutes and
centrifuged for 5 minutes at 2000 RPM. The supernatant is
discarded, and the pellet is dried under vacuum. The pellet is
dissolved in 3.3 mL of H.sub.2O, and to the resulting solution is
added a solution of 4.3 mg (0.005 mmol) of 36 in 2.0 mL of pH 8.0
100 mM sodium borate. To the resulting solution is added 250 uL (50
umol) of a 200 mM solution of pyridine-borane complex in MeOH, and
the mixture is kept at 37.degree. for 4 days. The conjugate, E, can
be purified by ion exchange chromatography.
Combination 4: Activated Carboxylate on Platform--Amine on
Ligand
##STR00018##
[0206] Compound F--Platform with Four Carboxylic Acid Groups.
Succinic anhydride (1.0 g, 10 mmol) is added to a solution of 861
mg (1.0 mmol) of 36 and 252 mg (3.0 mmol) of NaHCO.sub.3 in 20 mL
of 1/1 dioxane/H.sub.2O, and the mixture is stirred for 16 h at
room temperature. The mixture is acidified with 1 N HCl and
concentrated. The concentrate is purified by silica gel
chromatography to provide F.
[0207] Compound G--Platform with Four N-Succinimidyl Esters. A
solution of 126 mg (0.1 mmol) of F and 46 mg (0.4 mmol) of
N-hydroxysuccinimide in 5 mL of anhydrous THF is prepared. The
mixture is cooled to 0.degree. and 103 mg (0.5 mmol) of
dicyclohexylcarbodiimide is added. The mixture is stirred allowing
to come to room temperature over several hours. The solids are
removed by filtration, and the filtrate is concentrated to provide
G which can be purified by silica gel chromatography.
[0208] Compound Z--Peptide with Amino Group. A peptide is
synthesized with standard solid phase methods on a Wang
(p-alkoxybenzyl) resin. Lysine c-amines are protected as CBZ
groups. Amino acid residues are added sequentially to the amino
terminus using FMOC chemistry. The last residue added is
N-FMOC-aminocaproic acid. After cleaving from the resin with
trifluoroacetic acid, the FMOC group is removed with piperidine to
provide a peptide with a free amine linker. The peptide, Z, is
purified by reverse phase HPLC.
[0209] Peptide--Platform Conjugate H. A solution of 0.05 mmol of Z
and 0.1 mmol of Et.sub.3N in 1 mL of DMF is prepared. To the
solution is added a solution of 16.5 mg (0.01 mmol) of G in 1 mL of
DMF. The mixture is stirred until the reaction is complete. To
remove protecting groups, the conjugate is dissolved in MeOH, and
the solution is placed in a Parr hydrogenation apparatus with 100
mg of 10% Pd/C per gram of conjugate. The mixture is shaken under
60 psi H.sub.2, and the deprotected conjugate, H, is purified by
preparative reverse phase HPLC.
Combination 5: Isothiocyanate on Platform--Amine on Ligand
##STR00019##
[0211] Compound 1--Platform with Four Isothiocyanates. Thiophosgene
(381 uL, 575 mg, 5.0 mmol) is added to a solution of 861 mg (1.0
mmol) of 36 in 10 mL of THF, and the mixture is stirred at room
temperature until complete by TLC. The mixture is partitioned
between methylene chloride and a solution of 5% NaHCO.sub.3. The
extracts are dried (MgSO.sub.4), filtered, and concentrated. The
product, I, is purified by silica gel chromatography.
[0212] Peptide--Platform Conjugate, J. A solution of 0.05 mmol of Z
and 0.1 mmol of Et.sub.3N in 1 mL of DMF is prepared. To the
solution is added a solution of 10.3 mg (0.01 mmol) of I in 1 mL of
DMF. The mixture is stirred until the reaction is complete. To
remove protecting groups, the conjugate is dissolved in MeOH, and
the solution is placed in a Parr hydrogenation apparatus with 100
mg of 10% Pd/C per gram of conjugate. The mixture is shaken under
60 psi H.sub.2, and the deprotected conjugate, J, is purified by
preparative reverse phase HPLC.
Combination 6: Chloroformate on Platform--Amine on Ligand
##STR00020##
[0214] Compound K--Platform with Four Hydroxyl Groups. A solution
of 205 uL (275 mg, 1 mmol) of triethylene glycol bis-chloroformate
in 5 mL of CH.sub.2Cl.sub.2 is added to a solution of 497 uL (525
mg, 5 mmol) of diethanolamine and 696 uL (506 mg, 5 mmol) of
Et.sub.3N in 5 mL of CH.sub.2Cl.sub.2 at 0.degree.. The mixture is
allowed to warm to room temperature and stirred until complete as
evidenced by TLC. The mixture is concentrated and the product, K,
is isolated by silica gel chromatography.
[0215] Compound L--Platform with Chloroformate Groups. Pyridine
(100 ul) followed by 1.19 g (4 mmol) of triphosgene are added to a
solution of 412 mg (1 mmol) of K in 20 mL of CH.sub.2Cl.sub.2. The
mixture was stirred at room temperature for 20 hours, and the
solvent was evaporated under vacuum to give compound L.
[0216] Peptide--Platform Conjugate, M. A solution of 1 mmol of Z in
10 mL of pyridine is added to a solution of 132 mg (0.2 mmol) of L
in 5 mL of 1/1 THF/pyridine. The mixture is stirred until the
reaction is complete. Solvents are removed in vacuo. To remove
protecting groups, the conjugate is dissolved in MeOH, and the
solution is placed in a Parr hydrogenation apparatus with 100 mg of
10% Pd/C per gram of conjugate. The mixture is shaken under 60 psi
H.sub.2, and the deprotected conjugate, M, is purified by
preparative reverse phase HPLC.
EXAMPLE 4
Synthesis of Conjugates Comprising Two Different Biological
Molecules
[0217] It can be useful to conjugate more than one kind of
biologically active group to a platform molecule. This example
describes the preparation of a platform containing two maleimide
groups, which react with a thiol-containing peptide, and two
activated ester groups, which react with a drug containing a free
amine. The resulting conjugate contains two peptides and two drug
molecules as shown in Scheme 20.
[0218] Preparation of heteroactivated valency platform molecule
Benzyl 6-aminocaproate tosylate salt, K: A mixture of 32 mmol of
6-aminocaproic acid, 51 mmol of p-toluenesulfonic acid, and 40 mmol
of benzyl alcohol in 60 mL of toluene is refluxed using a
Dean-Stark trap to remove water. When the reaction is complete, the
mixture is cooled, and the product precipitates. The solid is
collected by filtration and recrystallized from EtOH/Et.sub.2O to
provide compound K.
[0219] Compound L: Dicyclohexylcarbodiimide (2 equivalents) is
added to a solution of 1 equivalent of compound 5 and 2 equivalents
of N-hydroxysuccinimide in THF. The mixture is stirred for 4 hours
and 2.2 equivalents of compound K is added. The mixture is stirred
until the reaction is complete as evidenced by TLC. The mixture is
filtered and concentrated. The product is purified by silica gel
chromatography.
[0220] Compound M: Compound L is treated with trifluoroacetic acid
in CH.sub.2Cl.sub.2. When the reaction is complete, the mixture is
concentrated under vacuum to provide compound M as the
trifluoroacetate salt.
[0221] Compound N: Compound 6 (Scheme 2) is treated with
trifluoroacetic acid in CH.sub.2Cl.sub.2. When the reaction is
complete, the mixture is concentrated under vacuum to provide
compound N as the trifluoroacetate salt.
[0222] Compound O: 3.2 mmol of triethyleneglycol bis-chloroformate
is added to a solution of 4 mmol of compound M and 4 mmol of
compound N in 162 mL of pyridine in a 20.degree. water bath. The
mixture is stirred until complete by TLC and concentrated under
vacuum. The concentrate is dissolved in CH.sub.2Cl.sub.2 and washed
successively with 1 N HCl solution, 5% NaHCO.sub.3 solution, and
saturated NaCl solution. The CH.sub.2Cl.sub.2 layer is dried
(MgSO.sub.4), filtered and concentrated. The concentrate is
dissolved in 10 mL of EtOH and 10 mL of 1 M NaOH is added. The
mixture is stirred for several hours, until no further reaction
appears to take place by TLC. The mixture is acidified to pH 1 with
1 N HCl and extracted with CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2
layer is dried (MgSO.sub.4), filtered, and concentrated. The
product, O is isolated by silica gel chromatography.
[0223] Compound P: Compound O is dissolved in EtOH and hydrogenated
in a Parr shaker with 100 mg of 10% palladium on carbon per gram of
O. The reaction is monitored for completeness by TLC. When the
reaction is complete, the catalyst is removed by filtration, and
the mixture is concentrated to yield compound P.
[0224] Compound Q: 3 mmol of N-methoxycarbonylmaleimide is added to
a solution of 1 mmol of compound P in 20 mL of dioxane and 5 mL of
saturated NaHCO.sub.3 at 0.degree.. The mixture is stirred for an
hour, acidified with 1 N HCl, and extracted with CH.sub.2Cl.sub.2.
The CH.sub.2Cl.sub.2 layer is dried (MgSO.sub.4), filtered, and
concentrated, and the product is purified by silica gel
chromatography to yield Q.
[0225] Compound R: 2 mmol of DCC is added to a solution of 1 mmol
of Q and 2 mmol of p-nitrophenol in CH.sub.2Cl.sub.2 and the
mixture is stirred for 16 h. The solids are removed by filtration,
and the filtrate is concentrated and purified by silica gel
chromatography to yield R.
[0226] Conjugate with two peptides and two drug molecules, compound
S: An excess of two equivalent of thiol-containing peptide is added
to a solution of 1 equivalent of heteroactivated platform, R, in pH
7.5 phosphate buffer. The mixture is stirred for 1 hour, and excess
of two equivalents of amine-containing drug is added. The
conjugate, S, is isolated by reverse-phase HPLC or ion-exchange
chromatography or a combination of both.
##STR00021## ##STR00022##
EXAMPLE 5
Synthesis and Testing of Conjugate 3-II
##STR00023##
[0227] Preparation of DMTr-5'-Modified (CA).sub.10.
[0228] The polynucleotide d-[DMTr-(bzCp(CE)bzA).sub.10] was
prepared on a Milligen 8800 Prep Scale DNA synthesizer (See FIG.
6A) following the manufacturer's protocols for DNA phosphoramidite
synthesis. The synthesis was carried out on 10 g of DMTr-d-bzA-CPG
support with a nucleoside loading of 30.0 .mu.mol/g. The final DMTr
blocking group was removed using the machine protocol. Milligen
activator solution, Cat. No. MBS 5040 (45 mL) and 0.385 g of
compound 51 (see Reaction Scheme 11) were added to the reaction and
the suspension was mixed for 8 minutes by argon ebullition. The
mixture was oxidized by the usual machine protocol and the
support-bound polynucleotide was collected by filtration, air dried
and treated with 100 mL of concentrated ammonia for 16 hours at
55.degree. C. When cool, the mixture was filtered through a Gelman
10 .mu.m polypropylene filter. The filter was washed with 200 mL of
2 mM NaCl adjusted to pH 12 with NaOH. The filtrate was then
applied to an Amicon chromatography column (0.45.times.9.4 cm, 150
mL) which had been packed with Q-Sepharose (Pharmacia, Peapack,
N.J.) equilibrated first with 3M NaCl and then with 2 mM NaCl, pH
12. The column was eluted with 500 mL of a linear gradient (2 mM
NaCl, pH 12 to 1.3 M NaCl, pH 12), then washed with 1.3 M NaCl, pH
12 until all U.V. absorbing material came off. Fractions which
absorbed at 260 nm were further analyzed by polyacrylamide
electrophoresis and those containing pure product were pooled. The
pool (120 mL) was treated with 240 mL of cold isopropanol and
stored for 30 minutes at -20.degree. C. The precipitate was
collected by centrifugation in a Sorvall RC 3B centrifuge using a
model H-6000A rotor for 15 minutes at 3000 rpm and 4.degree. C. to
yield DMTr-5'-modified (CA).sub.10 (14946 A.sub.260 units, 498 mg,
62.2 .mu.Mol, 20% based on 300 .mu.Mol CPG nucleoside.)
Synthesis of a Tr-5'-Modified (CA).sub.10
##STR00024##
[0230] The synthesis of Tr-5'-modified (CA).sub.10 was carried out
as described above for the synthesis of DMTr-5'-modified
(CA).sub.10 (prepared as described in Reaction Scheme 11) by
substituting compound 53 for compound 51.
Conjugation of DMTr-5'-Modified Polynucleotides to Compound 3
(IA-DABA-PEG, Reaction Scheme 1)--Preparation of Conjugate 3-I
[0231] In the conjugation procedures that follow, all the buffers
and solutions employed were thoroughly sparged with helium and all
reaction vessels were purged with argon before use. A solution of
11,568 A.sub.260 units (48.2 .mu.mol, assume molar extinction at
260 nm=240,000) of the DMTr-5'-modified (CA).sub.10 in 7.7 mL water
was treated with 1 mL of 0.1 M NaHCO.sub.3 and 210 .mu.L (876
.mu.mol, 18 times molar excess) tributylphosphine for 0.5 hour at
room temperature. The suspension was shaken from time to time. The
suspension was treated with 0.8 mL of 3M NaCl and 16 mL of cold
isopropanol. After 30 minutes at -20.degree. C., the material was
centrifuged at 3000 rpm for 20 minutes. The pellet was redissolved
in 2 mL of water, 0.2 mL of 3M NaCl, treated with 4 mL isopropanol
and recentrifuged. The pellet was briefly dried under vacuum and
dissolved in 2.8 mL of water and 1 mL of 0.1 N NaHCO.sub.3 which
had been sparged with helium. 6.7 mg of compound 3 (IA-DABA-PEG)
was added, and the mixture was kept for 16 hours at room
temperature in the dark. The reaction mixture in a final volume of
6 mL was applied to a 5.times.91 (1800 Ml) Pharmacia column which
was packed with Sephacryl 200 (Pharmacia). The column was eluted
with 0.5 M NaCl, 0.1 M sodium borate, pH 8.3. A peristaltic pump
was used and set to give a flow rate of approximately 2 mL per
min., and fractions of 15 ml were collected. The absorbance of the
fractions at 260 nm was measured. The fractions were also analyzed
by polyacrylamide gel electrophoresis and those containing pure
conjugate were pooled.
Hybridization of Conjugate 3-1--Preparation of Conjugate 3-II
[0232] The pooled fractions from above contained 726 A.sub.260
units. The equivalent amount of (TG).sub.10 was added and the tube
was heated at 90.degree. C. for ten minutes and then allowed to
cool to room temperature over 1.5 hours. An equal amount of
isopropanol was added and the mixture kept for 3 hours at
-20.degree. C. After centrifugation at 3000 rpm for 20 minutes, the
pellet was dissolved in 0.15 M NaCl, 0.01 M sodium citrate, pH 6.8.
53 mg of the hybrid was obtained. An aliquot of the material was
diluted in the above buffer and the melting temperature of the
duplex was determined in a Carey 3E spectrophotometer. The material
had a Tm of 73.4.degree. C. and 24.3% hyperchromicity. A 10
A.sub.260 unit aliquot of the product was annealed with excess
(TG).sub.10 as described above. This as well as unannealed
conjugate and a (TG).sub.10 standard were analyzed by gel
permeation HPLC on a Shodex Protein KW 8025 column on a Rainin HPLC
instrument. The column was eluted isocratically with 0.05M
NaH.sub.2PO.sub.4, pH 6.5, 0.5M NaCl. The run time was 12 minutes.
The product had a retention time of 6.9 minutes and (TG).sub.10 9.2
minutes. Comparison of the area under the peaks showed that 98.09%
of the product was double stranded DNA. The conjugate is
represented by the structure designated "Conjugate 3-II" in FIG.
6A.
EXAMPLE 6
Preparation of PN-KLH Conjugate
[0233] The PN-KLH conjugate was prepared according to the scheme
below:
##STR00025##
Synthesis of ACT-Modified (CA).sub.25
[0234] Compound 58 was coupled to (CA).sub.25 as the final step of
automated synthesis which incorporates the elements of an acyclic
triol moiety (ACT). Forty-nine sequential steps were carried out
using alternating dC and dA phosphoramidites beginning with 10 g of
DMT-d-bzA-CPG support with a nucleoside loading of 30 .mu.mol/g.
The DMTr blocking group was removed from the resulting
d-[DMTr-(BzCp(CE)BzA).sub.25], and 40 mL of activator solution
(Milligen, Cat. No. MBS 5040) and 800 mg of compound 58 were added
to the reaction mixture. The suspension was mixed for 8 minutes by
argon ebullition and subjected to a conventional oxidation step.
The support bound polynucleotide was removed from the reaction
vessel, air dried, and treated with 100 mL of concentrated ammonia
for 40 hours at 55.degree. C. When cool, the mixture was filtered
through a Gelman 10 .mu.m polypropylene filter and the filtrate was
then purified by conventional ion exchange chromatography.
Fractions which absorbed at 260 nm were further analyzed by
polyacrylamide gel electrophoresis and those containing pure
product were combined and precipitated with isopropanol to provide
510 mg (31.9 .mu.mol, 10%) of the ACT-modified (CA).sub.25.
Synthesis of Single-Stranded PN-KLH Conjugate
[0235] To a solution of 100 mg (2.5 .mu.mol) of NAIO.sub.4-treated
ACT-modified (CA).sub.25 in 1.33 mL of 50 mM sodium borate pH 8.0
was added 31.3 mg (0.208 .mu.mol) of KLH and 2.0 mg (31.8 .mu.mol)
of pyridine borane. The mixture was kept at 37.degree. C. for 72 h,
and the product was purified by chromatography on S-200.
Hybridization of Single-stranded PN-KLH Conjugate with
(TG).sub.25
[0236] The equivalent amount of (TG).sub.25 was added to the
single-stranded PN-KLH conjugate and the tube was heated at
90.degree. C. for ten minutes and then allowed to cool to room
temperature over an hour and a half. Precipitation with isopropyl
alcohol yielded 53 mg of product, PN-KLH; Tm (0.15 M NaCl, 0.01 M
sodium citrate, pH 6.8) 73.4.degree., 31.1% hyperchromicity; 98%
double stranded as determined by HPLC comparison to standards
consisting of sample annealed with excess (TG).sub.10, unannealed
conjugate, and unannealed (TG).sub.10 (Shodex Protein KW 8025
column, 0.05 M NaH.sub.2PO.sub.4, pH 6.5, 0.5 M NaCl). This
conjugate may be represented by the formula
KLH--[NH(CH.sub.2).sub.5OPO.sub.2O--(CA).sub.25:(TG).sub.25].sub..about.-
5
(assuming a molecular weight of 10.sup.5 for KLH) and is designated
"PN-KLH."
Testing of Conjugate 3-II as a Tolerogen
[0237] Conjugate 3-II was tested for its ability to tolerize mice
that had been immunized with an immunogenic form of the
polynucleotide, PN-KLH.
Material and Methods
[0238] Mice: C57BL/6 female mice 6 weeks of age were purchased-from
Jackson Laboratories, Bar Harbor, Me. The mice were housed and
cared for by NIH approved methods.
[0239] Immunization: The mice were primed, according to the method
of Iverson (Assay for in vivo Adoptive Immune Response in Handbook
of Experimental Immunology, Vol. 2 Cellular Immunology, Eds. D. M.
Weir, L. A. Herzenberg, C. Blackwell and A. Herzenberg, 4th
Edition, Blackwell Scientific Publications, Oxford) by injecting
the mice, i.p., with 100 .mu.g of PN-KLH precipitated on alum and
with 2.times.10.sup.9 formalin fixed pertussis organisms as an
adjuvant. The mice were boosted with 50 .mu.g of PN-KLH, in saline,
i.p.
[0240] Coupling of PN to SRBC: Sheep Red Blood Cells (SRBC) in
Alsevers were purchased from Colorado Serum Co., Denver, Colo., and
used within two weeks. The SRBC were coated with
(CA).sub.25:(TG).sub.25 (a 50 mer of CA:GT) by the method of Kipp
and Miller ("Preparation of Protein-Conjugated Red Blood Cells with
ECDI (Modification)" in Selected Methods in Cellular Immunology,
(1980), Eds. B. B. Mishell and S. M. Shiigi, W.H. Freemen and Co.,
San Francisco, p. 103). Briefly, the SRBC were washed 4 times in
cold saline, mixed with 2 mg of (CA).sub.25:(TG).sub.25 coupled to
D-EK in 0.35M mannitol, 0.01 M NaCl containing 10 mg of
carbodiimide and incubated for 30 minutes at 4.degree. C. The
coated SRBC were washed twice with cold Balanced Salt Solution and
resuspended to 10% (v/v).
[0241] Plaque assay: The number of anti-PN plaque forming cells
(pfc) was determined using the Cunningham technique (Marbrook, J.,
"Liquid Matrix (Slide Method)", in Selected Methods in Cellular
Immunology, (1980), Eds. B. B. Mishell and S. M. Shiigi, W.H.
Freemen and Co., San Francisco, p. 86.). The number of IgG pfc were
determined by elimination of IgM plaques using rabbit and
anti-mouse IgG as described by Henry ("Estimation of IgG responses
by Elimination of IgM Plaques" in Selected Methods in Cellular
Immunology, (1980), Eds. B. B. Mishell and S. M. Shiigi, W.H.
Freemen and Co., San Francisco, p. 91). Briefly, spleens were
harvested and single cell suspensions made in balanced salt
solution (BSS). Guinea pig serum was added to polynucleotide coated
SRBC to give a final dilution of 1:9 guinea pig serum, and enough
rabbit anti-mouse IgG was added to give a final dilution of 1:100
rabbit anti-mouse IgG. Equal volumes of the SRBC mixture and
diluted spleen cells were mixed in microtiter wells and transferred
to Cunningham chambers. Each spleen was tested individually and in
triplicate. The edges of the chambers were sealed with paraffin and
the chambers were incubated at 37.degree. C. for 1 hour. The number
of plaques were enumerated by viewing the chambers under an
inverted microscope.
Results
[0242] Mice were primed with PN-KLH precipitated on alum with
pertussis as an adjuvant (A&P) and seven weeks later divided
into groups of 3 mice each. The mice were treated, i.p., with
doubling dilutions of PN-DABA-PEG, Conjugate 3-II five days later
all of the mice, including the control, were boosted with 50 .mu.g
of PN-KLH, in saline, i.p. Four days later, the spleens were
harvested and the number of IgG pfc determined. As shown in Table
2, all doses of Conjugate 3-II tested showed a significant
reduction in the number of pfc as compared to the control
group.
TABLE-US-00002 TABLE 2 Tolerogenic Activity of Conjugate 3-II
(PN-DABA-PEG) Dose pfc/10.sup.6 spleen cells % Reduction
(.mu.g/mouse) Mean (S.D.) Mean None 12865 (2846) 62.5 2868 (6809)
77.7 125 3331 (939) 74.1 250 3044 (1929) 76.3 500 1809 (759) 85.9
1000 2814 (554) 78.1
EXAMPLE 7
Preparation and Testing-of Conjugate 20-II
Conjugation of Tr-5' Modified (CA).sub.10 to Valency Platform
Molecule 20--Preparation of Single-Stranded Conjugate 20-I
[0243] 969 .mu.L (789 mg, 3.89 mmol) of tri-n-butylphosphine was
added to a solution of 918 mg (0.14 mmol) of Tr-5'-modified
(CA).sub.10 in 30 mL of H.sub.2O under argon atmosphere. The
mixture was stirred for 1 hour and then 2.4 mL of a 3M NaCl
solution was added followed by 42 mL of isopropanol which had been
sparged with helium to remove oxygen. The mixture was placed in a
freezer at -20.degree. C. for 1 hour and then centrifuged at 3000
rpm for 30 minutes. The supernatant was removed and the oily
residue was dissolved in 15.5 mL of helium sparged H.sub.2O. 1.24
mL of 3M NaCl and 21.7 mL of helium sparged isopropanol was added
to the mixture. The resulting mixture was then placed in a freezer
at -20.degree. C. for 1 hour and centrifuged at 3000 rpm for 20
minutes. The oily pellet was dried under vacuum for 18 hours to
yield a solid. The solid was dissolved in 6 mL of helium sparged
H.sub.2O to give a total volume of 6.4 mL. The amount of DNA was
863 mg as determined by UV absorbance at 260 nm (0.033 mg per unit
of absorbance in pH 7.5 phosphate buffered saline). The solution
was transferred to a 50 mL three-neck flask under argon. One neck
of the flask had an argon gas inlet while the other two necks were
stoppered. The total volume was adjusted to 7.7 mL with H.sub.2O
and 0.87 mL of helium sparged 1M sodium phosphate buffer, pH 7.8,
and 0.97 mL of MeOH. 1.9 mL (33.63 mg, 0.025 mmol) of a 17.7 mg/mL
solution of compound 20 in MeOH was added to the mixture. The
resulting mixture was stirred under argon for 20 hours and then
diluted to 100 mL with a solution comprising 0.1 M NaCl, 0.05 M
sodium phosphate, pH 7.5, and 10% MeOH. Purification was
accomplished by chromatography on Fractogel.RTM. (equilibration:
0.1 M NaCl, 0.05 M sodium phosphate, pH 7.5, 10% MeOH: elution
gradient 0.5 M NaCl, 0.05 M sodium phosphate, pH 7.5, 10% MeOH to
0.8 NaCl, 0.05 M sodium phosphate, pH 7.5, 10% MeOH). Fractions
containing pure conjugate 20-I as evidenced by HPLC and
polyacrylamide gel electrophoresis were collected in 232 mL of
eluent. The product and salts were precipitated by adding an equal
volume of isopropanol and placing same in a freezer at -20.degree.
C. for 1 hour. Dialysis against H.sub.2O (2.times.100 vol) gave 335
mg of conjugate 20-I (32 mL of 10.47 mg/mL, 0.033 mg/absorbance
unit at 260 .mu.m, assumed).
Annealing of Conjugate 20-I with (TG).sub.10 to Form
Double-Stranded Conjugate 20-II
[0244] 150 mg (14.33 mL of 10.47 mg/mL based on 0.033 mg/absorbance
unit at 260 nm) of conjugate 20-I and 157.5 mg (1.50 mL of 104.6
mg/mL based on 0.033 mg/absorbance unit at 260 nm) of (TG).sub.10
were placed into a 50 mL polypropylene centrifuge tube. The
concentration was adjusted to 15 mg/mL by adding 2.0 mL of pH 7.2
10.times.PBS and 2.17 mL of H.sub.2O. The mixture was placed in a
90.degree. C. water bath and allowed to cool to room temperature
over 1.5 hours. The concentration was determined to be 17.7 mg/mL
by absorbance at 260 nm (0.050 mg/absorbance unit); transition melt
temperature 67.5.degree. C.; hyperchromicity 27%; osmolality 346;
pH 7.2. For final formulation of conjugate 20-II, the solution was
diluted to a final concentration of 12.7 mg/mL and an osmolality of
299 by adding 7.23 mL of pH 7.2.times.PBS and filtering through a
0.22 L filter.
Alternative Conjugation of Tr-5'-Modified (CA).sub.10-20,
Preparation of Single Stranded Conjugate 20-I
[0245] 10 equivalents of tri-n-butylphosphine are added to a 10
mg/mL solution of Tr-5'-modified (CA).sub.10 in He sparged with 100
mM pH 5 sodium acetate. The mixture is stirred for 1 hour and then
precipitated with 1.4 volumes of isopropyl alcohol (IPA). The
mixture is placed in the freezer at -20.degree. C. for 1 hour and
centrifuged at 3000 rpm for 20 minutes. The supernatant is removed
and the pellet is dissolved to 10 mg/mL in He sparged IPA. The
mixture is placed in the freezer at -20.degree. C. for 1 hour and
centrifuged at 3000 rpm for 20 minutes. The pellet is dried under
vacuum for 18 hours to give a solid. A 50 mg/mL solution of the
solid is prepared in He sparged 100 mM pH 10 sodium borate buffer.
0.25 equivalents of compound 20 as a 40 mg/mL solution in 9/1
MeOH/H.sub.2O is added to the mixture. The mixture is stirred at
room temperature for 3-20 hours and diluted (0.1 M NaCl, 0.05
sodium phosphate, pH 7.5, 10% MeOH). Purification is accomplished
by chromatography on Fractogel.RTM. (equilibration; 0.1 M NaCl,
0.05 M sodium phosphate, pH 7.5, 10% MeOH: elution gradient; 0.5 M
NaCl, 0.05 M sodium phosphate, pH 7.5, 10% MeOH to 0.8 M NaCl, 0.05
sodium phosphate, pH 7.5, 10% MeOH). Fractions containing pure
20-I, as evidenced by HPLC and polyacrylamide gel electrophoresis,
were collected. The product and salts are precipitated by adding an
equal volume of IPA and standing in the freezer at -20.degree. C.
for 1 hour. Dialysis against H.sub.2O (2.times.10 vol) give
20-I.
Alternative Annealing of 20-I with (TG).sub.10-20 to Form Double
Stranded Conjugate 20-II
[0246] The methodology is essentially the same as that described
above except that annealing is done at 70.degree. C. instead of
90.degree. C.
Second Alternative Conjugation of Tr-5'-Modified (CA).sub.10-20,
Preparation of Single Stranded Conjugate 20-I
[0247] 4.8 mL of tri-n-butylphosphine was added to a solution of
7.75 g of Tr-5'-modified (CA).sub.10 in 104 mL of Ar sparged 100 mM
pH 5 sodium acetate under N.sub.2. The mixture was stirred for 1
hour and then precipitated with 232.5 mL of IPA. The mixture was
placed in a freezer for -20.degree. C. for 1.5 hours, centrifuged
at 3000 rpm for 20 minutes and then frozen at -20.degree. C. for 24
hours. The supernatant was removed and the pellet was dissolved in
170 mL He sparged 0.3 M NaCl solution. The mixture was again
precipitated with 232 mL of Ar sparged IPA. The mixture was then
placed in a freezer at -20.degree. C. for 2 hours, centrifuged at
3000 rpm for 20 minutes and then frozen at -20.degree. C. for 11
hours. The supernatant was decanted and the pellet was dried under
vacuum for 12 hours to give a solid. A solution of the solid was
prepared in 110 mL of Ar sparged 100 mM pH 10 sodium borate buffer.
406 mg of compound 20 as a solution in 4.4 mL of 9/1 MeOH/H.sub.2O
was added to the mixture. The mixture was stirred at room
temperature for 2 hours. The product mixture contained 62% of 20-I
by high-pressure ion chromatography, Waters Gen Pak Fax column
(100.times.4 mm), 60.degree. C., linear gradient from 65% A/35% B
to 18% A/82% B; A=0.05 M NaH.sub.2PO.sub.4, pH 7.5, 1 mM EDTA, 10%
MeOH (v/v); B=0.05 M NaH.sub.2PO.sub.4, pH 7.5, 1 M NaCl, 1 mM
EDTA, 10% MeOH (v/v), eluting at 19.5 minutes.
Testing of Conjugate 20-II and Nonconjugated Controls
[0248] C57BL/6 mice were immunized with PN-KLH and A&P. After
three weeks, groups of 5 mice/group were treated with either
different doses of Conjugate 20-II or 4.5 nM HAD-AHAB-TEG (linker,
HAD, attached to derivatized valency platform molecule, AHAB-TEG,
see FIG. 7), or 18 nM (4.times.4.5) (CA).sub.10:(TG).sub.10, or a
mixture of 4.5 nM HAD-AHAB-TEG plus 18 nM (CA).sub.10:(TG).sub.10,
i.p.; and one group was not treated. The groups were given booster
injections and the sera were collected and assayed as described in
Example 6. The percent reduction of the anti-PN response is shown
in FIG. 4. The anti-KLH responses of these mice was normal and were
not significantly different than those shown in FIG. 2. The results
clearly show that the anti-PN response was not affected by (i) the
valency platform molecule alone, (ii) the PN alone, or (iii) a
mixture of the two. The PN must be coupled to the nonimmunogenic
valency platform molecule in order to induce tolerance.
Conjugate 20-II Causes a Reduction in the Number of PN-specific
Antibody Producing Cells
[0249] C57Bl/6 mice were immunized with PN-KLH, A&P. After
three weeks, groups of 3 mice/group were treated with different
doses of Conjugate 20-II, i.p; one group was not treated. After
five days, all of the mice were given a booster injection of PN-KLH
in saline, i.p., and then 4 days later their spleens were harvested
and assayed for the number of PN-specific, IgG-producing cells
using the hemolytic plaque assay. The results, shown in Table 3,
clearly show that this conjugate reduced the number of PN-specific
IgG-producing cells.
TABLE-US-00003 TABLE 3 REDUCTION IN THE NUMBER OF pfc BY Conjugate
20-II PN-specific pfc per Dose 10.sup.6 spleen cells Group#
.mu.g/mouse (Mean & S.E.) Reduction 1 None 5562 (2570) 2 274
982 (1871) 82.3 3 91 1867 (1335) 66.4 4 30 2247 (1606) 59.6 5 10
6109 (2545) 0 6 3 4045 (1411) 27.3 7 1 4578 (2475) 17.7 8 0.4 5930
(897) 0
EXAMPLE 8
Testing of Conjugates as Tolerogens
Testing of Conjugate 17-II as a Tolerogen
[0250] C57BL/6 mice were immunized with PN-KLH, A&P. Three
weeks later groups of 5 mice/group were treated with different
doses of Conjugate 17-II intraperitoneally, (i.p.), and one group
was not treated. Five days later all of the mice were given a
booster injection of PN-KLH, in saline, i.p., and 7 days later the
mice were bled. The sera were analyzed for anti-PN antibody by the
Farr assay at a PN concentration of 10.sup.-8M. The percentage
reduction of the anti-PN response is shown in FIG. 1. The sera were
also analyzed for anti-KLH antibodies using an ELISA assay. The
results, expressed as the percentage of anti-KLH compared to a
standard pool of anti-KLH sera, are shown in FIG. 2. The data in
FIG. 1 show that this conjugate reduces the anti-PN response. The
anti-KLH (platform molecule) response in all of the mice is normal
(see FIG. 2).
Testing of Various of the 11 Series of Conjugates as Tolerogens
[0251] Groups of three C57BL/6 mice/group were immunized with
PN-KLH, A&P. After three weeks, two groups were treated with 3
different doses of either Conjugate 11-IV, Conjugate 11-II,
Conjugate 11-VI or Conjugate 11-VIII, i.p., one group was not
treated. These conjugates are described in FIGS. 6A-B and were
prepared according to the methodology described above in Example 7.
Five days later all of the mice were given a booster injection of
PN-KLH, in saline, i.p., and 7 days later the mice were bled. The
sera were analyzed for anti-PN antibody by the Farr assay at a PN
concentration of 10.sup.-8M. The results showing the percentage
reduction in the anti-PN response are presented in FIG. 3. The
anti-KLH responses in these mice were not significantly different
than the responses shown in FIG. 2. All four conjugates
significantly reduced the anti-PN response at all doses tested.
Conjugate 11-II Causes a Reduction in the Number of PN-specific
Antibody Producing Cells
[0252] C57Bl/6 mice were immunized with PN-KLH, A&P. After
three weeks, groups of 3 mice/group were treated with different
doses of Conjugate 11-II, i.p., one group was not treated. Five
days later, all of the mice were given booster injections of PN-KLH
in saline, i.p.; and 4 days later their spleens were harvested and
assayed for the number of PN-specific, IgG-producing cells using
the hemolytic plaque assay. The results of this experiment with
different doses of Conjugate 11-II are shown in Table 4. These
results clearly show that this conjugate reduced the number of
PN-specific IgG-producing and that the reduction in antibody titer
was not due to the clearance of serum antibody bound to
conjugate.
TABLE-US-00004 TABLE 4 REDUCTION IN THE NUMBER OF pfc BY Conjugate
11-II PN-specific pfc per 10.sup.6 spleen cells % Reduction Group#
.mu.g/mouse (Mean & S.E.) (SD) 1 None 10845 (1308) 2 263 3613
(547) 66.23 (8.6) 3 87 3462 (1041) 64.98 (17) 4 29 7354 (1504) 29.5
(23.8) 5 9 6845 (2031) 30.9 (32.2) 6 3 7982 (223) 26.8 (3.52) 7 1
6043 (545) 44.5 (7) 8 0.4 9343 (1251) 13 (19.8)
EXAMPLE 9
Preparation of HADpS-(CA).sub.10-Conjugate 20-IV
[0253] A modified polynucleotide with a phosphorothioate joining
the linker to the 5' end was prepared. Synthesis of the twentymer,
(CA).sub.10, and the addition of the HAD linker to the
polynucleotide was carried out according to the methodology of
Example 5 except for the following. In the final oxidation step,
the iodine solution was replaced with a 0.05 M solution of
3H-1,2-benzodithiole-3-one 1,1 dioxide (Glen Research, Sterling,
Va.) in acetonitrile. Sulfurization was carried out according to
the manufacturer's instruction. Ammonia treatment and purification
were carried out as in Example 5. Conjugation of the polynucleotide
to the AHAB-TEG valency platform were carried out according to the
methodology of Example 5.
Testing of Conjugate 20-IV as a Tolerogen
[0254] Because the 5' phosphate on the PN may be susceptible to
enzymatic degradation, one of the oxygen molecules on the terminal
phosphate was replaced with sulfur--thus the name HADPS. C57BL/6
mice were immunized with PN-KLH, A&P. After three weeks, groups
of 5 mice/group were treated with different doses of Conjugate
20-IV, i.p.; one group was not treated. The groups were given
booster injections and the sera were collected and assayed as
described above. The results showing the percentage reduction in
the anti-PN response are shown in FIG. 5. The anti-KLH responses of
these mice (data not shown) were normal and were not significantly
different that those shown in FIG. 2. These results show that this
conjugate significantly reduced the anti-PN response.
Conjugate 20-IV Causes a Reduction in the Number of PN-specific
Antibody Producing Cells
[0255] C57Bl/6 mice were immunized with PN-KLH, A&P. After
three weeks, groups of 3 mice/group were treated with different
doses of Conjugate 20-IV, i.p.; one group was not treated. After
five days, all of the mice were given a booster injection of PN-KLH
in saline, i.p., and 4 days later their spleens were harvested and
assayed for the number of PN-specific, IgG-producing cells using
the hemolytic plaque assay. The results, shown in Table 5, show
that this conjugate reduced the number of PN-specific IgG-producing
cells.
TABLE-US-00005 TABLE 5 REDUCTION IN THE NUMBER OF pfc BY Conjugate
20-IV PN-specific pfc per Dose 10.sup.6 spleen cells Group#
.mu.g/mouse (Mean & S.E.) % Reduction 1 None 5889.4 (3444) 2
274 3413 (1604) 42 3 91 222 (752) 96.2 4 30 1492 (2269) 74.7 5 10
5421 (832) 8 6 3 5077 (1946) 13.9 7 1 7023 (679) 0 8 0.4 4159
(2688) 29
EXAMPLE 10
Treatment of BXSB Mice With LJP 394, Conjugate 20-II
Mice Treatment Protocol
[0256] Six to 9 week old male BXSB mice (Jackson Laboratory, Bar
Harbor, Me.) were housed at the La Jolla Pharmaceutical facility.
Food and water were provided as libitum. Animals were rested one
week prior to use. Initial blood samples and weights were obtained
prior to the first conjugate treatment. Conjugate treatment was
initiated at 7 to 9 weeks of age and was administered intravenously
twice weekly from day 59 to day 150. Animals were bled periodically
and their anti-DNA antibody titers were determined.
Assay for IgG Anti-DNA Antibody Production
[0257] A serum sample taken from each mouse was assessed for the
presence of anti-DNA antibody by ELISA. Falcon Probind 96 well
microtitration assay plates (Becton Dickerson, Oxnard, Calif.) were
coated with 100 .mu.L/well of (PN).sub.50-D-EK (a co-polymer of
D-glutamic acid and D-lysine) at a concentration of 50 .mu.g/mL
overnight at 4.degree. C. The plates were washed twice with PBS
without calcium and magnesium and 0.05% Tween 20 (wash buffer)
using a M96V plate washer (ICN Biomedical, Inc., Irvine, Calif.).
Plates were blocked for 1 hour at room temperature in PBS
containing 1% gelatin (Norland Products, Inc., New Brunswick, N.J.)
and 0.05% Tween 20. Plates were washed twice with wash buffer
before the addition of serum samples or standards. Serum samples
and standards were prepared in a diluent containing PBS with 1%
gelatin, 0.05% Tween 20 and 10% goat serum. Plates were incubated
with serum samples for 60 to 90 minutes at 37.degree. C. and then
the wells were washed four times with wash buffer. Biotinylated
goat anti-mouse IgG (Sigma Chemical Co., St. Louis, Mo.) was
diluted 1/1000 in blocking solution containing 10% goat serum. The
plates were incubated for 1 hour at 37.degree. C. and washed four
times. The substrate, OPD (Sigma Chemical Co., St. Louis, Mo.), was
added. The plates were incubated in the dark until the highest
reading of the highest standard was approximately 1 OD unit by an
ELISA plate reader at OD 450 nm (Bio-Tek Instruments, Winooski,
Vt.). The reaction was stopped with 50 .mu.L of 3M HCl and the
plates were read at 490 nm. The reference positive serum was
included in each microtitration plate and the positive wells from
each assay were within the sensitivity range of the reference curve
95% of the time. In the later bleeds, some positive samples
exceeded the reference curve. However the most dilute mouse serum
sample was within the range of the reference curve. No significant
binding was observed by normal control negative serum. The results
are shown in FIG. 15.
EXAMPLE 11
Preparation of Melittin Peptides and Conjugate
[0258] The melittin molecule, composed of 26 amino acids, is one of
the major components of bee venom. One third of the bee venom
sensitive individuals have melittin specific antibodies. Melittin
is highly immunogenic in some mouse strains (Balb/c, CAF1). The
majority (>80%) of melittin specific antibodies in the responder
mouse strains bind a B cell epitope which is the c-terminal
heptapeptide of melittin.
TABLE-US-00006 Melittin (SEQ. ID.: 1)
H.sub.2N-Gly-Ile-Gly-Ala-Val-Leu-Lys-Val-Leu-Thr-Thr-
Gly-Leu-Pro-Ala-Leu-Ile-Ser-Trp-Ile-Lys-Arg-Lys-
Arg-Gln-Gln-CONH.sub.2. Melittin Peptides for T cell Stimulation
Melittin Peptide #1. (SEQ. ID NO.: 2)
Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly ("7 mer"). Melittin Peptide #2.
(SEQ. ID NO.: 3) Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly ("8 mer").
Melittin Peptide #3. (SEQ ID NO.: 4)
Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly ("9 mer"). Melittin Peptide
#4. (SEQ. ID NO.: 5) Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly
("10 mer"). Melittin Peptide #5. (SEQ. ID NO.: 6)
Cys-Ile-Ser-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly ("11 mer").
Peptide Synthesis
[0259] Melittin peptides were synthesized using standard Fmoc
chemistry techniques on a glycine resin (Advanced ChemTech #SG5130
or equivalent (Advanced ChemTech, 2500 Seventh Street Road,
Louisville, Ky.) using 2.3 M excess amino acid derivatives for each
coupling step. Completion of the coupling was monitored with
bromphenol blue and confirmed with ninhydrin.
TABLE-US-00007 Melittin Peptides Used in Conjugations Melittin
Peptide #6. (SEQ. ID NO.: 7)
H.sub.2N-Cys-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Gly-CO.sub.2H.
Melittin Peptide #7. (SEQ. ID NO.: 8)
H.sub.2N-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln-Lys-Cys-Gly- CO.sub.2H.
Melittin Peptide #8. (SEQ. ID NO.: 10)
(H.sub.2N-Trp-Ile-Lys-Arg-Lys-Arg-Gln-Gln).sub.2-Lys-Cys-
Gly-CO.sub.2H.
[0260] A cysteine was added as required for coupling certain
peptides via a thioether bond to the valency platform molecule.
Peptides were purified by reversed phase HPLC following synthesis
and lyophilized to dryness. The appropriate amount of peptide was
then weighed out for each conjugation.
Reduction of Preformed Disulfide Bonds: (Tributylphosphine
Method)
[0261] All buffers were sparged with helium. The peptide was
dissolved in a minimal volume (approximately 10 to 20 mg/mL) of
0.05 M NaHCO.sub.3 (pH 8.25). A 1 mL solution of 0.7 M
tributylphosphine (TBP; MW=202.32 g/mole; d -0.812 g/mL) was
prepared by adding 174.4 .mu.L of TBP to 825.6 .mu.L of isopropanol
(iPrOH). Then, 1:1 equivalents of TBP were added to the peptide
solution prepared as described above, mixed well, and allowed to
react for 30 minutes to 1 hour with occasional mixing to keep TBP
dissolved and/or dispersed in the solution. Complete reduction was
confirmed by HPLC.
Conjugation of Peptides to Valency Platform Molecule #3 or #60:
[0262] All buffers were sparged with helium. The polyethylene
glycol (PEG) derivative #3 or #60 was dissolved in a minimal volume
(approximately 20 mg/mL) of 0.05 M NaHCO.sub.3 (pH 8.25).
Approximately 3 equivalents of peptide were used per iodacetyl
group on the PEG derivative. For para-aminobenzoic acid (PABA)-PEG;
2 iodacetyl groups; MW=approximately 4100 g/mole; 6 equivalents of
peptide were used for each equivalent of PABA-PEG. For
diaminobenzoic acid (DABA)-PEG; 4 iodoacetyl groups;
MW=approximately 4300 g/mole; 12 equivalents of peptide were used
for each equivalent of DABA-PEG. The PEG solution was added to the
reduced peptide solution and allowed to react for at least one hour
in the dark. The peptide conjugate was purified by preparative
HPLC. Before pooling and lyophilization, fractions were checked by
electrophoresis using a 15% tricine gel.
TABLE-US-00008 TABLE 6 Conjugates of Melittin Peptides and PEG T
cell Valence # B cell activation Conjugate plat- Peptide epitopes/
Conjugation by peptide or number form conjugated molecule terminus
conjugate.sup.1 1 60 6 2 N no (pep) 2 3 6 4 N no (pep/conj) 3 3 7 4
C nd 4 3 5 4 N yes (pep) 5 3 9 .sup. 8.sup.2 C nd .sup.1Stimulation
of uptake of [.sup.3H] thymidine by cultured T cell from
melittin-immunized mice; nd = not determined; pep = peptide tested;
conj = peptide-PEG conjugate tested. .sup.24 copies of a branched
peptide, containing two identical branches each; each branch
comprising a B cell epitope
Murine Lymph Node Proliferation Assays.
[0263] Female Balb/c mice (6-8 weeks old; Jackson Laboratory, Bar
Harbor, Me.) were obtained and housed at the La Jolla
Pharmaceutical animal facility according to National Institutes of
Health guidelines. Food and water was provided ad libitum. Balb/c
mice were immunized in each hind footpad with 50 .mu.g of melittin
molecule in Complete Freund's Adjuvant (CFA) (Sigma Chemical Co.,
St. Louis, Mo.). Popliteal lymph nodes were harvested aseptically
seven days later. Lymph nodes were gently dissociated by teasing
the cells through a 50 mesh sieve screen. The single cell
suspension was washed in RPMI-1640 (Irvine Scientific, Irvine
Calif.) containing glutamine, penicillin and streptomycin.
5.times.10.sup.5 cells in RPMI medium supplemented with 10% fetal
bovine serum (FCS) in quadruplicate wells of round bottom 96-well
Corning microtitration plates were cultured with melittin or a
melittin peptide at 10, 1.0 or 0.1 .mu.g/mL. Cells in the positive
control wells were cultured with murine interleukin 2 (IL-2) at 100
or 50 U/mL, PHA (phytohemagglutinin) at 1 .mu.g/mL. The negative
control wells contained lymph node cells in RPM-1640 and 10% FCS.
The cells were cultured for 4 days in a 37.degree. C. incubator
with 5% CO.sub.2. Each well was pulsed with 1 .mu.Ci of
[.sup.3H]thymidine (ICN Biochemicals, Costa Mesa, Calif.) for an
additional 18 hours. Cells were harvested onto a glass fiber filter
mat using a semiautomatic cell harvester (Scatron, Sterling, Va.).
Incorporation of [.sup.3H]thymidine was determined by liquid
scintillation. The results were expressed as average counts per
minute.
In Vivo Protocols
[0264] Balb/c mice were primed intraperitoneally (i.p.) with 4
.mu.g of melittin in CFA. One month later the potential tolerogen
or formulation buffer was administered i.p. Three days later all
mice received an i.p. injection of 4 .mu.g of melittin in
Incomplete Freund's Adjuvant (ICF) (Sigma Chemical Co., St. Louis,
Mo.). 100 to 200 .mu.L of blood was collected from the
retro-orbital venous plexus 10 days later. Serum samples were
assayed for anti-peptide or anti-melittin IgG antibodies.
Assay for IgG Anti-Melittin or Total Anti-Melittin Antibodies
[0265] An individual mouse's serum sample was assessed serially for
the presence of anti-melittin antibodies by ELISA. Falcon Probind
96-well microtitration plates were precoated with 10 .mu.g/mL
melittin or melittin peptide in phosphate buffered saline (PBS), pH
7.2, overnight at 4.degree.. The plates were washed twice with a
wash solution containing PBS, 0.02% Tween-20, and 1% gelatin
(Norland Products Inc., New Brunswick, N.J.). Plates were blocked
with 200 .mu.L PBS containing 5% gelatin for 1 hour at 37.degree..
Serum samples were prepared in a diluent of PBS containing 5%
gelatin. Samples were tested at dilutions of 1:100 to 1:1000. After
1 hour of incubation at 37.degree. C., the plates were washed four
times. ExtraAvidin peroxidase (Sigma Chemical Co., St. Louis, Mo.)
was diluted 1:1000 in PBS containing 5% gelatin. The plates were
incubated 30 minutes at 37.degree. C. and then washed five times.
Wells were developed with o-phenylenediamine (OPD) (Sigma Chemical
Co., St. Louis, Mo.) in the dark for 15-30 minutes, the reaction
was stopped with 3 M HCl. The optical density (OD) was determined
at 450 nm on a microplate reader (Bio-tek Instruments, Winooski,
Vt.).
Antibody Forming Cell Assay
[0266] Cellulose microtitration plates (Millipore Co., Bedford,
Mass.) were prepared as indicated above for the IgG antibody
(ELISA) assay. However, at the point in the assay where the serum
samples were added to the wells, splenic cells
(5.times.10.sup.5/well) were added instead of serum, and incubated
overnight. The remainder of the ELISA assay was performed as
indicated above.
T Cell Epitopes
[0267] T Cells from mice primed with melittin showed T cell
proliferation in response to the whole melittin molecule and to
C-terminal melittin peptides 3, 4, and 5 (FIG. 8). However,
C-terminal peptides 1 and 2 induced no significant T cell
proliferation. Melittin peptides 2 and 5 were conjugated to PEG.
Like melittin peptide 2, the PEG conjugate of melittin peptide 2
also did not induce significant T cell proliferation.
Studies Using Melittin Conjugated Peptides to Tolerize Mice Primed
and Boosted with Melittin
[0268] Mice treated with the conjugate prepared as described above
(10 mg/kg, 200 .mu.g/mouse), had significantly lower levels of
anti-melittin peptide 2 antibodies (FIG. 9) and also lower levels
of anti-melittin antibodies (FIG. 10) as compared to the control
Balb/c mice treated with formulation buffer. Spleen cells from mice
treated with buffer control or the conjugate were assayed for the
ability of antibody-forming cells to produce anti-melittin or
anti-melittin peptide 2 antibodies as measured in a soluble ELISA
assay. As shown in FIG. 11, the levels of anti-melittin peptide 2
antibody forming cells in the conjugate treatment group were
significantly lower than in the control group which was
administered formulation buffer. Mice treated with Conjugate 4, a
conjugate of peptide 5 which contains a T cell epitope, failed to
reduce the titer of antibodies to peptide 5 in treated mice. Thus,
the conjugate containing a T cell epitope was not a tolerogen (FIG.
12). In fact, rather than reduce the response, the levels of
anti-peptide antibody may have increased slightly.
EXAMPLE 12
Additional Studies Using Melittin Peptide Conjugates to Tolerize
Mice Primed and Boosted with Melittin
[0269] Female C57BL/6 mice, ages 5 to 8 weeks were purchased from
The Jackson Laboratory, Bar Harbor, Me. Animals were maintained and
treated accordingly to National Institutes of Health
guidelines.
Immunization Protocol
[0270] Mice were primed by an i.p. injection containing 5 .mu.g of
melittin precipitated on alum and 2.times.10.sup.9 B. pertussis
(Michigan Department of Public Health, Lansing, Mich.) as an
adjuvant. The mice were boosted with 5 .mu.g of melittin, i.p., in
PBS.
pfc Assay
[0271] Sheep Red Blood Cells (SRBC) (Colorado Serum Co., Denver,
Colo.) were conjugated with melittin-peptide 2 using carbodiimide.
Fresh SRBC (less than 2 weeks old) were washed four times with cold
saline and one time with mannitol (0.35 M mannitol, 0.01 M NaCl).
The SRBC were suspended in mannitol to a concentration of 10%
(v/v). 100 .mu.L of mannitol containing 30 .mu.g of melittin
peptide #3 were added to 1 mL aliquots of 10% SRBC which were then
incubated on ice for 10 minutes. 100 .mu.L of a 100 mg/mL solution
of 1-ethyl-3 (3-dimethylaminopropyl)-carbodiimide HCl (EDCI) was
then added and incubated on ice for 30 minutes. The SRBC were
washed twice with Balanced Salt Solution (BSS) (Irvine Scientific
Co, Irvine, Calif.) and resuspended to 10% (v/v). Lyophilized
guinea pig complement (GIBCO, New York, N.Y.) was reconstituted
with BSS and then diluted 1:3 with BSS. One mL of the diluted
guinea pig complement was added to 3 mL of conjugated SRBC. Rabbit
anti-mouse IgG was added to give a final dilution of 1:100 of the
rabbit antiserum. This concentration was predetermined to inhibit
all IgM pfc while enhancing the maximum number of IgG pfc. An equal
volume of this complement/anti-mouse IgG/SRBC suspension was mixed
with a cell suspension of mouse spleen cells taken from a single
mouse. 50 .mu.L of each mixture was transferred to the chambers of
a Cunningham slide (three chambers per slide). The edges were then
sealed with paraffin and incubated at 37.degree. C. for one hour.
The number of plaques per chamber was counted with the aid of a
dissecting microscope. Each spleen suspension was also assayed
using non-conjugated SRBC as a control. The number of viable cells,
in each spleen cell suspension, was determined. The number of pfc
per 106 spleen cells was determined for each chamber and the mean
of the triplicates calculated. The number of pfc for non-conjugated
SRBC was subtracted from the number of pfc for conjugated SRBC to
determine the number of peptide-specific pfc.
Determining The Optimal Time to Measure pfc
[0272] Mice were primed with melittin. Groups (3 mice per group) of
primed mice were boosted with melittin on days 2, 4, 6, and 8. On
day 10 the mice were sacrificed and their spleens harvested. Cell
suspensions were prepared and assayed for the number of peptide
specific pfc determined. The optimal number of pfc was obtained 6
days after boosting with melittin.
The Orientation of the Peptide on the PEG Conjugate Does Not Affect
the Conjugate's Ability to Induce Tolerance
[0273] Two different tolerogens were constructed to determine if
the orientation of the peptide on the PEG conjugate affects its
ability to induce tolerance. The peptide was covalently bound to
valency platform molecule 3 through its C-terminal end to make
melittin conjugate 3. Groups (3/group) of mice primed with melittin
were treated, i.p., with conjugates or with saline. Five days later
all of the mice, including the non-treated control group, were
boosted with 5 .mu.p of melittin. Six days later the mice were
sacrificed, their spleens were harvested and the number of peptide
specific pfc determined. As illustrated in Table 8, both
orientations were effective in reducing the number of
peptide-specific pfc/10.sup.6 spleen cells in mice primed and
boosted with the parent protein Melittin.
TABLE-US-00009 TABLE 7 Orientation of the peptide on the PEG
conjugate does not affect the conjugates' ability to induce
tolerance Peptide specific pfc Melittin per 10.sup.6 spleen cells
Conjugate# .mu.g/mouse (Mean and S.D.) % Reduction 3 1000 .mu.g 386
(85) 86.8% '' 500 .mu.g 489 (one mouse) 83.3% '' 250 .mu.g 957
(298) 67.3% 2 1000 .mu.g 546 (160) 81.3% '' 500 .mu.g 866.6 (235)
70.4% '' 250 .mu.g 1280 (one mouse) 56.2% None None 2924 (164)
--
The Number of Peptides Per PEG Conjugate Does Affect the
Conjugate's Ability to Induce Tolerance
[0274] Three different conjugates, each with a different number of
peptides per PEG conjugate, were constructed to determine if the
ratio of peptides to PEG molecule was important. Conjugate 1 had
only two peptides per PEG conjugate. Another had four peptides per
PEG conjugate (Conjugate 2). The third had eight peptides per PEG
conjugate (Conjugate 5). Groups (3/group) of mice primed with
melittin were treated, i.p., with the different conjugates or with
saline. Five days later all of the mice, including the non-treated
control group, were boosted with 5 .mu.g of melittin. Six days
later, the mice were sacrificed, their spleens were harvested and
the number of peptide-specific pfc determined. As shown in Table 8,
Conjugate 1, containing two peptides per PEG molecule, was
ineffective in reducing the number of peptide-specific pfc/106
spleen cells in mice primed and boosted with the parent protein
melittin. The results show that both melittin conjugates 2 and 5
were effective as tolerogens; however, conjugate 5, which contained
8 peptides, was effective at a lower dose than conjugate 2 which
contained four peptides per valency platform molecule.
TABLE-US-00010 TABLE 8 The number of peptides per PEG conjugate
does affect the conjugates' ability to induce tolerance Treatment
Dose .mu.g/mouse Peptide specific Molecule (nMoles) indirect IgG
pfc (SD) % Reduction No treatment 1159 (280) std Conjugate 1 1000
(217) 1290 (98) -11% 250 (54) 1350 (206) -16% Conjugate 2 500 (80)
585 (125) 49.5% 250 (40) 1001 (176) 14% Conjugate 5 500 (53) 630
(325) 45.6% 250 (26.5) 443 (105) 61.8% 125 (13.25) 583 (69)
49.7%
[0275] Modifications of the above-described modes for carrying out
the invention that are obvious to those of skill in the fields of
polynucleotide chemistry, conjugation chemistry, immunology and
related fields are intended to be within the scope of the following
claims.
* * * * *