U.S. patent application number 13/433373 was filed with the patent office on 2012-10-11 for diarylsulfide backbone containing photolabile protecting groups.
This patent application is currently assigned to NIMBLEGEN SYSTEMS GMBH. Invention is credited to Klaus-Peter Stengele.
Application Number | 20120258891 13/433373 |
Document ID | / |
Family ID | 43927643 |
Filed Date | 2012-10-11 |
United States Patent
Application |
20120258891 |
Kind Code |
A1 |
Stengele; Klaus-Peter |
October 11, 2012 |
DIARYLSULFIDE BACKBONE CONTAINING PHOTOLABILE PROTECTING GROUPS
Abstract
The present disclosure relates to photoactivable protecting
groups containing a diarylsulfide chromophore, a method for the
synthesis thereof and their use as photoactivable protecting groups
using maskless photolithography based array synthesis.
Inventors: |
Stengele; Klaus-Peter;
(Pleiskirchen, DE) |
Assignee: |
NIMBLEGEN SYSTEMS GMBH
Waldkreiburg
DE
|
Family ID: |
43927643 |
Appl. No.: |
13/433373 |
Filed: |
March 29, 2012 |
Current U.S.
Class: |
506/32 ;
536/26.7; 536/26.8; 536/27.3; 536/28.51; 536/28.54; 548/375.1;
548/531; 558/282; 560/160 |
Current CPC
Class: |
C07D 213/70 20130101;
C07H 19/06 20130101; C07D 207/16 20130101; C07H 19/16 20130101;
C07K 7/08 20130101; C07D 235/28 20130101; C07H 19/20 20130101; C07H
19/10 20130101; C07C 323/32 20130101; Y02P 20/55 20151101; C07C
319/14 20130101; C07D 231/12 20130101; C07C 323/19 20130101; C07H
1/00 20130101; C07H 19/02 20130101; C07K 1/04 20130101 |
Class at
Publication: |
506/32 ; 558/282;
560/160; 548/531; 548/375.1; 536/28.54; 536/26.8; 536/28.51;
536/27.3; 536/26.7 |
International
Class: |
C40B 50/18 20060101
C40B050/18; C07C 271/22 20060101 C07C271/22; C07H 19/173 20060101
C07H019/173; C07D 231/12 20060101 C07D231/12; C07H 19/073 20060101
C07H019/073; C07H 19/10 20060101 C07H019/10; C07C 69/96 20060101
C07C069/96; C07D 207/16 20060101 C07D207/16 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 7, 2011 |
EP |
11161556.3 |
Claims
1. A compound of the formula ##STR00019## wherein Y is S or O, and
A is selected from the group consisting of --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH(CH.sub.3)--CH.sub.2--, and R1 is an unsubstituted or
substituted aryl- or heteroaryl-group, and R3 is H, a methyl group
or an ethyl group, and wherein R2 is H, forms a phosphoramidite,
H-phosphonate or phosphate triester, or wherein R2 is ##STR00020##
or wherein R2 is ##STR00021## wherein R4 is H, forms a
phosphoramidite, H-phosphonate or phosphate triester, and wherein
R5 is H, OH, a halogen or XR6, wherein X is O or S and R6 is H, an
alkyl-group, aryl-group, or OR6 forms a phosphoramidite,
phosphodiester, phosphotriester, H-phosphonate or an acetal or
silicone moiety, and wherein B is selected from the group
consisting of adenine, cytosine, guanine, thymine, uracil,
2,6-diaminopurine-9-yl, hypoxanthin-9-yl, 5-methylcytosinyl-1-yl,
5-amino-4-imidazolecarboxylic acid-1-yl or
5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein when B is
adenine, cytosine or guanine the primary amino group optionally has
a protecting group or when B is thymine or uracil at the O.sup.4
position is optionally a protecting group, or wherein R.sub.2 is
##STR00022## wherein R7 is a natural amino acid, a non-natural
amino acid or an amino acid derivative forming an urethan bond to
formula Ib, or wherein formula IV represents the carboxy function
of a natural amino acid, a non-natural amino acid or an amino acid
derivative, forming an ester bond to formula Ib.
2. The compound according to claim 1 wherein R1 is a phenyl-group,
a tert-butyl-phenyl group, a 1- or 2-naphthyl-group, a
2-pyridyl-group an aminophenyl-group, an N-alkylaminophenyl-group,
an N-Acylaminophenyl-group, a carboxyphenyl-group, a
phenylcarboxylic ester or an amide.
3. The compound according to claim 1 wherein A is
--CH(CH.sub.3)--CH.sub.2--.
4. The compound according to claim 1 wherein R2 is a
phosphoramidite or --P(OCH.sub.2CH.sub.2CN)(N-iPr.sub.2).
5. The compound according to claim 1 wherein R3 is H or an ethyl
group.
6. The compound according to claim 1 wherein R4 is H and R5 is
H.
7. The compound according to claim 1 wherein B is selected from the
group consisting of adenine, cytosine, guanine, thymine or
uracil.
8. The compound according to claim 1 wherein when B is adenine,
cytosine or guanine the protecting group is phenoxyacetyl-,
4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or
dimethylformamidino-residues, when B is adenine the protecting
group is benzoyl- or
p-nitro-phenyl-ethoxy-carbonyl-(p-NPPOC)-residues, when B is
guanine the protecting group is isobutyroyl-, p-nitrophenylethyl
(p-NPE) or p-NPEOC-residues and when B is cytosine the protecting
group is benzoyl-, isobutyryl- or p-NPEOC-residues.
9. The compound according to claim 1 wherein R7 is a natural amino
acid.
10. A method for preparing a diarylsulfide backbone containing one
or more photolabile protecting group(s) according to claim 1
comprising the steps of a) Provision of p-diethylbenzene as a
starting material; b) Bromination of the phenylring; c) Nitration
of the obtained compound in Nitric- and Sulfuric Acid in the
position para- to the Bromine; d) Purification and crystallization;
e) Hydroxymethylation of the compound at the benzylic position; f)
Conversion of the aromatic bromine group to the arylsulfide using
thiophenol; g) Purification; h) Conversion of the alcohol to
chlorocarbonate; and i) Reaction of the chlorocarbonate with a
nucleoside and reaction of the nucleoside with a phosphitylating
agent, or Reaction of the chlorocarbonate with an amino acid
derivative.
11. The method according to claim 10, characterized in that R1 is a
phenyl-group, a tert-butyl-phenyl group, a 1- or 2-naphthyl-group
or a 2-pyridyl-group.
12. The method according to claim 10, characterized in that A is
--CH(CH.sub.3)--CH.sub.2--.
13. The method according to claim 10, characterized in that R3 is H
or an ethyl group.
14. A method of forming an array through photolithography, said
method comprising contacting a substrate having a site activated by
exposure to light with a compound of claim 1.
15. The method of claim 14 wherein R.sub.2 is ##STR00023## wherein
R7 is a natural amino acid, a non-natural amino acid or an amino
acid derivative forming an urethan bond to formula Ib, or wherein
formula IV represents the carboxy function of a natural amino acid,
a non-natural amino acid or an amino acid derivative, forming an
ester bond to formula Ib.
16. The method of claim 14 wherein the contacting of the substrate
with a compound of claim 1 produces a DNA microarray or a peptide
microarray.
17. The method of claim 16 wherein the method produces a DNA
microarray.
18. The method of claim 16 wherein the method produces a peptide
microarray.
19. The method of claim 14 wherein the substrate and/or the
compound is activated by exposure to light between the wavelengths
of 374 to 405 nm.
Description
PRIORITY CLAIM
[0001] This application claims the benefit of European Patent
Application No. 11161556.3, filed Apr. 7, 2011, the disclosure of
which is hereby incorporated by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure relates to photoactivable protecting
groups (PLPGs). More specifically, the present disclosure relates
to the use of PLPGs in biomolecule synthesis.
BACKGROUND OF THE DISCLOSURE
[0003] Photolabile protecting groups (PLPG) play an important role
in blocking functional groups present in nucleosides, nucleotides,
sugars and amino acids, which are used for the synthesis of
biomolecules, e.g. nucleic acids and their derivatives, proteins,
peptides and carbohydrates. Additionally, PLPG have the advantage
that deprotection of the protected functional group can be
performed simply via light exposure. Therefore, PLPG provide the
basis for the photolithography based spatially resolved synthesis
of oligonucleotides or peptides on solid supports. The major
advantage of this technique is that high resolution microarrays can
be produced. Such high resolution microarrays are of great
significance for the analysis of biomolecules in medicine and
pharmaceutical research, as they provide the possibility to perform
high throughput and cost-effective analysis of multiple samples on
a single array. Examples of PLPG used for oligonucleotide synthesis
include, but are not limited to,
.alpha.-methyl-6-nitropiperonyl-oxycarbonyl (MeNPOC), and
2-(2-nitrophenyl)-propoxycarbonyl (NPPOC). Examples of PLPG used
for photolithography based peptide synthesis are for example
nitroveratryloxycarbonyl (NVOC) and 2-nitrobenzyloxycarbonyl
(NBOC).
[0004] Previous PLPG synthesis used light at a wavelength of
approximately 365 nm or shorter for the deprotection of the
protected functional groups. Light sources which are suitable to
generate such wavelength, are e.g. mercury arc lamps, excimer
lasers, UV-LEDs and frequency multiplied solid-state lasers. Such
light sources are characterized by high purchase costs, provide
limited luminous power and have a short life-time leading to high
overall costs of operation. Since some of the above mentioned light
sources contain hazardous substances, e.g. mercury, and appropriate
actions to secure occupational safety and proper disposal are
necessary, further increasing the costs.
[0005] Optical devices used for the photolithography based
synthesis of oligonucleotides or peptides, such as micro mirror
devices (WO 03/065038), are primarily designed for the visible
wavelength range of approximately 380 to 780 nm. Such devices carry
an antireflective or protective antiscratch coating optimized for
transparency for the respective visible wavelength range. Thus, the
near UV wavelength of 365 nm used for the deprotection of the
functional groups protected with PLPG require optical devices which
are optimized for near UV wavelengths. Since most of the optical
devices are optimized for the use with visible light, such
optimization often comprises removing the coating intended for the
use with visible light from the optical devices and/or coating the
optical device with materials intended for use with near UV or UV
light.
[0006] Furthermore, many light sources produce a broad spectrum of
wavelengths from the UV- to the IR-range, both of which have
disadvantageous effects concerning the synthesis of biomolecules.
As a result DNA or peptide microarrays might be of low quality due
to undefined lengths of the synthesized DNA strands and peptides,
respectively.
SUMMARY OF THE DISCLOSURE
[0007] PLPG are presented herein which are suitable for the
deprotection of the functional groups using visible light.
Consequently, harmless and cost-effective light sources as well as
regular optical elements can be used for the photolithography based
oligonucleotide and peptide synthesis.
[0008] The compounds of the present disclosure may be used for a
variety of different applications. In one aspect, the disclosure is
directed to the use of the compounds as photoactivable protecting
groups using maskless photolithography. In one embodiment the
compounds are used for the maskless photolithography based DNA
array synthesis as intermediate or permanent OH-protecting group in
nucleoside derivatives at the 3'-OH end or the 5'-OH end. Further,
the compounds are useful for maskless photolithography based
peptide array synthesis as NH-protecting group in amino acids. In
another embodiment the compounds are useful for the maskless
photolithography based peptide array synthesis as COOH-protecting
group in amino acids and/or for the maskless photolithography based
synthesis of carbohydrates as OH-protecting group and/or for
orthogonal protecting group strategy as SH-protecting group. In
another embodiment the compounds are used for maskless
photolithography having a wavelength of 374 to 405 nm, for example
a wavelength of 390 nm.
[0009] In another aspect, the disclosure is directed to a method
for the synthesis of a diarylsulfide backbone containing
photolabile protecting group as described above comprising the
steps of
[0010] Provision of p-diethylbenzene as a starting material
[0011] Bromination of the phenylring
[0012] Nitration of the obtained compound in Nitric- and Sulfuric
Acid in the position para- to the Bromine
[0013] Purification and crystallization
[0014] Hydroxymethylation of the compound at the benzylic
position
[0015] Conversion of the aromatic bromine group to the arylsulfide
using thiophenol
[0016] Purification
[0017] Conversion of the alcohol to chlorocarbonate
[0018] Reaction of the chlorocarbonate with a nucleoside and
reaction of the nucleoside with a phosphitylating agent, or [0019]
a. Reaction of the chlorocarbonate with an amino acid
derivative.
BRIEF DESCRIPTION OF THE FIGURES
[0020] The features of this disclosure, and the manner of attaining
them, will become more apparent and the disclosure itself will be
better understood by reference to the following description of
embodiments of the disclosure taken in conjunction with the
accompanying drawing.
[0021] FIG. 1 demonstrates half-lives of examples of PLPG according
to the disclosure in various solvents used at a wavelength of 390
nm. Light exposure was performed for various time periods.
[0022] FIG. 2 demonstrates half-lives of examples of PLPG according
to the disclosure in various solvents used at a wavelength of 404
nm. Light exposure was performed for various time periods.
[0023] FIG. 3 demonstrates certain UV absorption characteristics of
PLPG.
[0024] FIG. 4 exemplifies synthesis pathways of
disulfide-PLPG-amino acids.
[0025] FIG. 5 exemplifies synthesis pathways of
disulfide-PLPG-nucleotides.
[0026] FIG. 6 exemplifies synthesis pathways of further PLPG
according to the disclosure.
[0027] FIG. 7 exemplifies an alternative synthesis pathway of PLPG
without further alkyl substituents.
[0028] FIG. 8 shows a microarray scan of a peptide array containing
the target sequence of an anti-V5 antibody synthesized according to
the disclosure using disulfide-PLPG-amino acids.
[0029] Although the drawings represent embodiments of the present
disclosure, the drawings are not necessarily to scale and certain
features may be exaggerated in order to better illustrate and
explain the present disclosure. The exemplifications set out herein
illustrate an exemplary embodiment of the disclosure, in one form,
and such exemplifications are not to be construed as limiting the
scope of the disclosure in any manner.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0030] The embodiments disclosed herein are not intended to be
exhaustive or limit the disclosure to the precise form disclosed in
the following detailed description. Rather, the embodiments are
chosen and described so that others skilled in the art may utilize
their teachings.
[0031] The following definitions are set forth to illustrate and
define the meaning and scope of various terms used to describe the
disclosure herein.
[0032] The term "unsubstituted" is used herein as known to the
expert skilled in the art and refers to a hydrocarbon chain which
fully consists of carbon and hydrogen.
[0033] The term "substituted" is used herein as known to the expert
skilled in the art and refers to a replacement of a chemical group
or substituent (typically H or OH) with a functional group, and
particularly contemplated functional groups include electrophilic
groups (e.g., C(O)--OR, C(X)--OH, etc.), nucleophilic (e.g., --NH2,
--OH, --SH, --NC, etc.), ionic groups (e.g., --NH3-), polar groups
(e.g., --OH), non-polar groups (e.g., aryl, alkyl, alkenyl,
alkynyl, etc.), and halogens (e.g., --F, --Cl), and combinations
thereof.
[0034] The term "protecting group" is used herein as known to the
expert skilled in the art and refers to a substituent, functional
group, ligand, or the like, which is bonded (e.g., via covalent
bond, ionic bond, or complex) to a potentially reactive functional
group and prevents the potentially reactive functional group from
reacting under certain reaction conditions. Potentially reactive
functional groups include, for example, amines, carboxylic acids,
alcohols, double bonds, and the like. Protecting groups according
to the disclosure are photo labile protecting groups, which
include, but are not limited to, 2-Nitrobenzyloxycarbonyl (NBOC),
2-nitrophenyl-ethyloxycarbonyl (NPEOC),
2-(3,4-methylenedioxy-2-nitrophenyl)-propyloxy-carbonyl (MeNPPOC),
2-(3,4-methylenedioxy-2-nitrophenyl)-oxycarbonyl (MeNPOC),
2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC),
dimethoxy-benzo-inylyl-oxycarbonyl (DMBOC),
2-(2-nitrophenyl)-ethylsulfonyl (NPES),
(2-nitrophenyl)-propylsulfonyl (NPPS), and the like.
[0035] The term "aryl" is used herein as known to the expert
skilled in the art and refers to an aromatic residue consisting
solely of hydrogen and carbon atoms, such as a phenyl (C6H5-),
naphthyl (C10H7-) pyrenyl- or anthracenyl (C14H9-) residue. The
aryl can be substituted or unsubstituted with e.g. alkyl groups,
such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or
tert-butyl, or alkoxy- such as methoxy-ethoxy- or isopropoxy- or
halogen atoms, such as bromide, chloride, or fluoride.
[0036] The term "heteroaryl" is used herein as known to the expert
skilled in the art and refers to a cyclic aromatic group having
five or six ring atoms wherein at least one ring atom is selected
from the group consisting of oxygen, sulfur, and nitrogen, and the
remaining ring atoms are carbon. The heteroaromatic ring may form a
fused heteroaromatic system together with other aryl- or
heteroaryl-rings such as benzothiophene, benzimidazole, pteridine
or alloxazine.
[0037] The term "alkyl" is used herein as known to the expert
skilled in the art and refers to a univalent residue consisting
only of carbon and hydrogen atoms. The alkyls form homologous
series with the general formula CnH2n+1. The alkyl can be a
straight or branched alkyl, for example the alkyl can be a
secondary alkyl which is branched with the central carbon atom
linked to two carbon residues or a tertiary alkyl which is branched
with the central carbon atom linked to three carbon residues.
[0038] The letter A in the group -A-O-- represents a "fragmentation
linker" comprising from 1 to 2 linearly, covalently connected atoms
such as methylene- or ethylene-. The term "fragmentation linker" is
used herein as known to the expert skilled in the art and relates
to a moiety which is used as a moiety in photochemistry that
effects the light-induced fission of the PLPG by transforming the
primary photoprocess into a chemical cleavage reaction.
Accordingly, in a first aspect the divalent group -A- refers to a
linking group which connects the functional group R2 with the
nitrophenyl-chromophore. In one embodiment the 1 to 2 atom chain of
the linking group A can be fully comprised of hydrogen and carbon
atoms in form of a substituted or unsubstituted, branched or
linear, saturated or unsaturated hydrocarbon chain.
[0039] The hydrocarbon chain can also be branched having one or
more alkyl groups, wherein the alkyl group can be methyl, ethyl,
n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl.
[0040] Such a hydrocarbon chain can also be substituted by e.g.
halogen atoms. Accordingly, from 1 hydrogen atom to 3 hydrogen
atoms of the respective hydrocarbon chain can be substituted
through e.g. halogen.
[0041] The term "branched" in context with the definition of the
term linking group is used herein as known to the expert skilled in
the art and refers to the presence of a side-chain at the main
chain of the molecule or moiety. Accordingly, a branched linking
group can be a hydrocarbon chain as defined above having one or
more alkyl groups as side chain, wherein the alkyl group is methyl,
ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl; in
some embodiments the alkyl group is a methyl or ethyl group. In the
branched hydrocarbon chain represented by A from one to all carbon
atoms can have one or more alkyl groups as defined above.
[0042] The term "saturated" in context with the definition of the
term linking group is used herein as known to the expert skilled in
the art and relates to a linking group in which all members of the
group are connected to the respective adjacent atom(s) through
single bonds. Accordingly, a saturated hydrocarbon chain is
represented by the formula --(CH2)n- with n being an integer
ranging from 1 to 2.
[0043] The term "functional group" is used herein as known to the
expert skilled in the art and refers to any of numerous
combinations of atoms that form parts of chemical molecules, that
undergo characteristic reactions themselves, and that in many cases
influence the reactivity of the remainder of the molecule. Typical
functional groups are hydroxyl, carboxyl, aldehyde, carbonyl,
amino, azide, alkynyl, thiol and nitril.
[0044] The term "solid support" is used herein as known to the
expert skilled in the art and refers to any insoluble and rigid or
semi-rigid inorganic or organic material, often having a large
surface area to which surface organic molecules can be attached
through bond formation or absorbed through electronic or static
interactions such as through bond formation through a functional
group.
[0045] The term "biomolecule" is used herein as known to the expert
skilled in the art and refers to any organic molecule that is
produced by a living organism or to any artificially produced
derivatives of such compounds, including large polymeric molecules
such as proteins, polysaccharides, carbohydrates, lipids, nucleic
acids and oligonucleotides as well as small molecules such as
primary metabolites, secondary metabolites, and natural
products.
[0046] The term "nucleic acid" is used herein as known to the
expert skilled in the art and refers to a macromolecule composed of
chains of monomeric nucleotides, wherein each nucleotide consists
of three components: a nitrogenous heterocyclic base, which is
either a purine or pyrimidine; a pentose sugar; and a phosphate
group.
[0047] The term "natural amino acid" is used herein as known to the
expert skilled in the art and refers to one of the 20 canonical
amino acids used for protein biosynthesis as well as all amino
acids which can be incorporated into proteins during translation
(including pyrrolysine and selenocysteine). The 20 canonical amino
acids include histidine, alanine, valine, glycine, leucine,
isoleucine, aspartic acid, glutamic acid, serine, glutamine,
asparagine, threonine, arginine, proline, phenylalanine, tyrosine,
tryptophan, cysteine, methionine and lysine.
[0048] The term "non-natural amino acid" is used herein as known to
the expert skilled in the art and refers to organic compounds that
are not among those encoded by the standard genetic code or
incorporated into proteins during translation. Furthermore, the
term "non-natural amino acid" refers to organic compounds that do
not occur naturally. Therefore, non-natural amino acids include
amino acids or analogs of amino acids, but are not limited to, the
D-isostereomers of amino acids, citrulline, homocitrulline,
homoarginine, hydroxyproline, homoproline, ornithine,
4-amino-phenylalanine, cyclohexylalanine, .alpha.-aminoisobutyric
acid, N-methyl-alanine, N-methyl-glycine, norleucine,
N-methyl-glutamic acid, tert-butylglycine, .alpha.-aminobutyric
acid, tert-butylalanine, 2-aminoisobutyric acid,
.alpha.-aminoisobutyric acid, 2-aminoindane-2-carboxylic acid,
selenomethionine, dehydroalanine, lanthionine, .gamma.-amino
butyric acid, and derivatives thereof wherein the amine nitrogen
has been mono- or di-alkylated.
[0049] The term "peptide" is used herein as known to the expert
skilled in the art and refers to organic compounds made of amino
acids arranged in a linear chain and joined together by peptide
bonds between the carboxyl and amino groups of adjacent amino acid
residues.
[0050] The term "amino group" is used herein as known to the expert
skilled in the art and refers to primary (--NH2), secondary
(--NHR1), or tertiary (--N R1 R2), and in cationic form, may be
quaternary (--N R1 R2 R3). Examples of amino groups include, but
are not limited to, --NH2, --NHCH3, --NHC(CH3)2, --N(CH3)2 and
--N(CH2CH3)2. Examples of cyclic amino groups include, but are not
limited to, aziridino, azetidino, pyrrolidino, piperidino,
piperazino, morpholino, and thiomorpholino.
[0051] The term "maskless photolithography" is used herein as known
to the expert skilled in the art and refers to a technique for the
synthesis of DNA- or peptide-microarrays without the use of
photographic masks. The maskless photolithography uses an array of
optical switching elements that are individually addressable and
operable under software control. Examples for such optical
switching elements are micro mirror devices. One example of a micro
mirror device is the Digital Light Processor (DLP) from Texas
Instruments, Inc.
[0052] In a first aspect, the disclosure is directed to photolabile
protecting groups containing a diarylsulfide chromophore having the
general formula:
##STR00001##
[0053] wherein Y is S or O, and
[0054] A is selected from the group consisting of --CH.sub.2--,
--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH(CH.sub.3)--CH.sub.2--, and R1 is an unsubstituted or
substituted aryl- or heteroaryl-group, and R3 is H, a methyl group
or an ethyl group, and wherein R2 is H, forms a phosphoramidite,
H-phosphonate or phosphate triester, or wherein R2 is
##STR00002##
[0055] or wherein R2 is
##STR00003##
[0056] wherein R4 is H, or OR4 forms a phosphoramidite,
H-phosphonate or phosphate triester and wherein R5 is H, OH, a
halogen or XR6, wherein X is O or S and R6 is H, an alkyl-group,
aryl-group, or OR6 forms a phosphoramidite, phosphodiester,
phosphotriester or H-phosphonate or an acetal or a silicone moiety,
and wherein B is selected from the group consisting of adenine,
cytosine, guanine, thymine, uracil, 2,6-diaminopurine-9-yl,
hypoxanthin-9-yl, 5-methylcytosinyl-1-yl,
5-amino-4-imidazolecarboxylic acid-1-yl or
5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein when B is
adenine, cytosine or guanine the primary amino group optionally has
a protecting group or when B is thymine or uracil at the O.sup.4
position is optionally a protecting group,
[0057] or wherein R2 is
##STR00004##
[0058] wherein R7 is a natural amino acid, a non-natural amino acid
or an amino acid derivative forming an urethan bond to formula Ib
or wherein formula IV represents the carboxy function of a natural
amino acid, a non-natural amino acid or an amino acid derivative,
forming an ester bond to formula Ib.
[0059] In some embodiments, R1 is a phenyl-group, a
tert-butyl-phenyl group, a 1- or 2-naphthyl-group, a
2-pyridyl-group an aminophenyl-group, an N-alkylaminophenyl-group,
an N-Acylaminophenyl-group, a carboxyphenyl-group, a
phenylcarboxylic ester or an amide, and/or A is
--CH(CH.sub.3)--CH.sub.2-- and/or R2 is a phosphoramidite or
--P(OCH.sub.2CH.sub.2CN)(N-iPr.sub.2) and/or R4 is H and/or R5 is H
and/or R7 is a natural amino acid. When B is selected from the
group consisting of adenine, cytosine, guanine, thymine or uracil,
or when B is adenine, cytosine or guanine the protecting group is
typically selected from phenoxyacetyl-,
4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or
dimethylformamidino-residues, when B is adenine the protecting
group is typically selected from benzoyl- or
p-nitro-phenyl-ethoxy-carbonyl-(p-NPPOC)-residues, when B is
guanine the protecting group is typically selected from
isobutyroyl-, p-nitrophenylethyl (p-NPE) or p-NPEOC-residues and
when B is cytosine the protecting group is typically selected from
benzoyl-, isobutyryl- or p-NPEOC-residues.
[0060] In some embodiments, R1 is a phenyl-group, a
tert-butyl-phenyl group, a 1- or 2-naphthyl-group, a
2-pyridyl-group, A is --CH(CH.sub.3)--CH.sub.2-- and R3 is H or an
ethyl group.
[0061] Certain embodiments of the present disclosure relate to
diarylsulfide chromophore containing PLPG which can be used for the
photolithography based oligonucleotide and peptide synthesis having
the structure
##STR00005##
[0062] wherein A is selected from the group consisting of --CH2-,
--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH(CH.sub.3)--CH.sub.2--, --CH2-CH(Alky,Aryl)- and
--CH(CH3)-CH(Alkyl, Aryl)-
[0063] R1 is an unsubstituted or substituted aryl- or
heteroaryl-group or a condensed aryl- or heteroaryl-group, and R3
is H, a methyl group or an ethyl group, and
[0064] wherein R2 is
##STR00006##
[0065] or wherein R2 is
##STR00007##
[0066] wherein R4 is H, an alkyl-group, aryl-group, or OR4 forms a
phosphoramidite, H-phosphonate or phosphate triester and
[0067] wherein R5 is H, OH, a halogen or XR6, wherein X is O or S
and R6 is an alkyl-group, aryl-group, or OR6 forms a
phosphitamide-group, phosphodiester, phosphotriester or
H-phosphonate or an acetal or a silicone moiety and
[0068] wherein B is selected from the group consisting of adenine,
cytosine, guanine, thymine, uracil, 2,6-diaminopurine-9-yl,
hypoxanthin-9-yl, 5-methylcytosinyl-1-yl,
5-amino-4-imidazolecarboxylic acid-1-yl or
5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein when B is
adenine, cytosine or guanine the primary amino group optionally has
a protecting group or when B is thymine or uracil at the O.sup.4
position is optionally a protecting group,
[0069] or wherein R2 is
##STR00008##
[0070] wherein R7 is a natural amino acid, a non-natural amino acid
or an amino acid derivative, including but not limited to .alpha.-
or .beta.-amino acids, forming an urethan bond to formula Ia,
[0071] or wherein formula IV represents the carboxy function of a
natural amino acid, a non-natural amino acid or an amino acid
derivative, forming an ester bond to formula Ia, including but not
limited to .alpha.- or .beta.-amino acids.
[0072] In another embodiment compounds according to formula Ia are
used, characterized in that R1 is a phenyl-group, a
tert-butyl-phenyl group, a 1- or 2-naphthyl-group or a 2- or
4-pyridyl-group, A is --CH(CH.sub.3)--CH.sub.2--, R4 is H and R5 is
H, R4 is H and R5 is OH or OSi(Alkyl3).
[0073] In another embodiment compounds according to formula Ia are
used, characterized in that B is selected from the group consisting
of adenine, cytosine, guanine, thymine, 5-methylcytosineor
uracil.
[0074] In another embodiment compounds according to formula Ia are
used, characterized in that, when B is adenine, cytosine or guanine
the protecting group is phenoxyacetyl-,
4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or
dimethylformamidino-residues, when B is adenine the protecting
group is a benzoyl-residue, when B is guanine the protecting group
is a isobutyroyl-residue and when B is cytosine the protecting
group is benzoyl- or isobutyroyl-residues.
[0075] In another embodiment compounds according to formula Ia are
used, characterized in that R7 is a natural amino acid.
[0076] Some examples of the diarylsulfide chromophore containing
PLPG which can be used for the photolithography based
oligonucleotide and peptide synthesis have the structures:
##STR00009##
[0077] The present disclosure further relates to the use of the
compounds according to formula Ia as photoactivable protecting
group using maskless photolithography. In one embodiment of the
disclosure micro mirror devices are used to perform a spatial
selective exposure of the oligonucleotide and peptide microarrays
to visible light in order to deprotect nucleotides and amino acids,
respectively, in the exposed areas during the synthesis process.
Deprotection of nucleotides and amino acids, respectively, lead to
the release of the next linkage site for the respective next
nucleotide or amino acid. The next nucleotide or amino acid which
should be coupled to the released linkage site within the specific
areas is simply added by its provision within a solvent plus an
activating reagent which is poured onto the array. This strategy is
repeated until oligonucleotides and oligopeptides, respectively, of
the desired lengths and design are obtained. Using this strategy it
is possible to produce highly dense microarrays of at least 10,000
features per cm.sup.2. This strategy may produce microarrays with
even higher densities in the range of 100,000 to 500,000 features
per cm.sup.2.
[0078] Various embodiments of the PLPG according to the disclosure
can be removed by using visible light in a range from 375 nm to 420
nm, in the range from 390 to 405 nm, or in the range from 390 nm
and 404 nm, respectively. These wavelengths can be generated using
light sources which are much less expensive as compared to light
sources necessary to perform deprotection in the near UV range at
approximately 365 nm. In some embodiments, solid state lasers
within the range from 375 nm to 420 nm, alternatively 390 nm and
404 nm, are used as light sources to remove the PLPG according to
the disclosure. In other embodiments, LEDs (light emitting diodes)
with sufficient emission within the range from 375 nm to 420 nm, or
390 nm and 404 nm, are used as light sources to remove the PLPG
according to the disclosure. LEDs are one example of light sources
that are low cost products as they are produced in high quantities,
e.g. for the use in Blu-ray Players, that are useful in conjunction
with the PLPG of the disclosure.
[0079] In further embodiments micro mirror devices are used which
are optimized for the use of visible light in the range of 375 nm
to 420 nm, or in the range of 390 to 410 nm, or in the range of 390
nm and 404 nm, respectively. In a further embodiment the coating of
the micro mirror devices remain on the devices in order to be used
with visible light. Devices that are used for UV- or near UV-light
have to be optimized for that purpose, i.e. the coating on the
micro mirror elements has to be removed by polishing.
[0080] In another embodiment, LCD displays or a beam splitter can
be used as virtual masks between the light source and the synthesis
area.
[0081] Photolithographic synthesis of the oligonucleotides and
peptides, respectively, can be performed on a support, such as a
solid support. The support can be made of any material known by the
skilled person used for such a purpose, such as plastic, silicon,
diamond carbon or glass. Examples of the material are optical grade
polyolefin or optical grade microscope glass slides. The support
can be provided in any form, such as beads, gels, plates,
membranes, slides or chips. The support can be transparent or
non-transparent, in various embodiments the support exhibits at
least 30%, at least 60%, at least 90% light transmission at a
wavelengths of between 375 nm to 410 nm.
[0082] The PLPG according to the disclosure can be used in any
process for oligonucleotide synthesis known by the skilled person
where protected nucleosides or nucleotides are necessary. The
PLPG-nucleotides as described herein can be used for the synthesis
of oligonucleotides in solution, further the PLPG-nucleotides as
described herein can be used for the synthesis of oligonucleotides
on a solid support. The synthesis can be performed by any standard
method known in the state of the art. In some examples the
synthesis can be performed by using photolithographic techniques,
such as maskless techniques wherein a micro mirror device is used
to expose light to spatial selected features on a microarray as
explained above.
[0083] Solvents known by the skilled person can be used during
oligonucleotide synthesis, such as acetonitrile.
[0084] Embodiments of the PLPG associated with nucleosides or
nucleotides for oligonucleotide synthesis can be used in a
concentration within the solvents of 1 mmol/L to 100 mmol/L, in a
concentration of 10 mmol/L to 40 mmol/L, or in a concentration of
25 mmol/L.
[0085] The PLPG used with nucleosides or nucleotides can be used in
connection with sensitizing agents known by the skilled person,
which increase the effectiveness of the deprotection reaction.
Examples of compounds useful as sensitizing agents are
benzophenone, xanthone and thioxanthone derivates, like e.g.
thioxanthen-9-one, alkylthioxanthen-9-ones, as for example
isopropylthioxanthen-9-one, 2-ethylthioxanthen-9-one,
2-chloro-thioxanthen-9-one, 1,4-dimethoxythioxanthen-9-one.
[0086] Oligonucleotide microarrays can be used for a variety of
purposes, including but not limited to sequence capturing,
comparative genomic hybridization (CGH), CHIP-chip analysis,
DNA-methylation analysis, gene expression analysis and comparative
genome sequencing.
[0087] In another embodiment compounds according to formula Ia are
used in the maskless photolithography based DNA array synthesis as
intermediate or permanent OH-protecting group in nucleoside
derivatives at the 3'-OH end or the 5'-OH end as carbon ester,
wherein the synthesis can be performed in 3'-5'-direction or in
5'-3'-direction.
[0088] If the PLPG is located at the 5'-end, the nucleotide carries
a phosphoramidite group on its 3'-end, which can be reacted with a
free --OH group on the solid support to form a stable elongated
oligonucleotide. After all oligonucleotides are synthesized, all
PLPG are removed and the oligonucleotide still bound to the solid
support has a free 5'-OH.
[0089] If however the PLPG is located at the 3'-end, the nucleotide
carries a phosphoramidite group on its 5'-end, which can be reacted
with any free --OH group on the solid support to form a stable
elongated oligonucleotide. After all oligonucleotides are
synthesized, all PLPG are removed and the oligonucleotide still
bound to the solid support has a free 3'-OH.
[0090] While both types of immobilization allow for hybridization
based assays, only the oligonucleotides that exhibit a free 3'-OH
may be used for enzymatic reactions for detection, labeling,
capping or elongation by ligation or enzymatic polymerization.
[0091] The PLPG according to the disclosure can further be used in
any process for peptide synthesis known by the skilled person where
protected amino acids are necessary. The amino acids can be
non-natural amino acids, amino acid derivatives and/or natural
amino acids. The PLPG as described herein can be used for the
synthesis of oligopeptides in solution, or the PLPG as described
herein can be used for the synthesis of oligopeptides on a solid
support. The synthesis can be performed by any standard method
known in the state of the art. For example, the synthesis can be
performed by using photolithographic techniques, such as techniques
where a micro mirror device is used to expose visible light to
spatial selected features on a microarray as explained above.
[0092] It has been shown that the deprotection reaction is
dependent on the solvent used during the peptide synthesis process.
Solvents known by the skilled person can be used during peptide
synthesis Polar solvents like dimethylsulfoxide (DMSO),
n-methylpyrrolidone (NMP), acetonitrile (MeCN) or isopropanol are
examples of solvents that can be used. Said solvents can contain
certain additives, such as imidazole, hydroxylamine and water.
Imidazole can be added at various concentrations, for example 0.1%
to 3% (v/v), 0.5% to 1.5% (v/v), or 1% (v/v). Hydroxylamine can be
added at concentrations of 0.1% to 3% (v/v), 0.2% to 1% (v/v), or
1% (v/v). Water can be added at concentrations of 0.1% to 20% (v/v
1% to 17% (v/v), or 1% (v/v). Examples of useful solvents are DMSO,
DMSO+1% imidazole, NMP+0.5% hydroxylamine, MeCN+1% H.sub.2O,
MeCN+1% H.sub.2O+1% imidazole, isopropanol+1% imidazole,
isopropanol+12% H.sub.2O+1% imidazole.
[0093] The PLPG associated to amino acids for peptide synthesis can
be used in a various concentrations within the solvents, such
concentrations such as 0.1 mmol/L to 0.5 mmol/L, 0.2 mmol/L to 0.4
mmol/L, or the PLPG can be used in a concentration of 0.3
mmol/L.
[0094] The PLPG associated to amino acids can be used in connection
with sensitizing agents known by the skilled person, which increase
the effectiveness of the deprotection reaction.
[0095] Oligopeptide microarrays can be used for a variety of
purposes, including but not limited to screening of antibody
libraries, quantitative or qualitative analysis of biological
samples, biomarker discovery, enrichment of scarce proteins,
depletion of high abundant proteins, analysis of
protein-protein-interactions, analysis of DNA-protein-interactions
or RNA-protein-interactions.
[0096] In another embodiment compounds according to formula Ia are
used for the maskless photolithography based peptide array
synthesis as NH-protecting group in amino acids as urethan. The
PLPG is used as NH-blocked free acid, activated ester, acid
halogenide, anhydride, intermolecular or intramolecular as
N-carboxy-anhydride (NCA).
[0097] In another embodiment the compounds according to formula Ia
are used for the maskless photolithography based peptide array
synthesis as COOH-protecting group in amino acids as ester for
inverse direction of synthesis.
[0098] The PLPG according to the disclosure can further be used in
any process known by the skilled person where protected sugars are
necessary. The sugars used can be compounds, such as aldohexoses
and aldopentoses. The PLPG as described herein can be used for the
synthesis of carbohydrates, glycoproteins and proteoglycans in
solution. In some embodiments, the PLPG as described herein can be
used for the synthesis of carbohydrates, glycoproteins and
proteoglycans on a solid support. The synthesis can be performed by
any standard method known in the state of the art. For example, the
synthesis can be performed by using photolithographic techniques,
such as techniques where a micro mirror device is used to expose
visible light to spatial selected features on a microarray as
explained above.
[0099] Carbohydrate microarrays can be used for a variety of
purposes, including but not limited to analysis of
saccharide-protein-interactions, high-throughput analysis of
proteins and cells, and analysis of glycans and their molecular
interactions.
[0100] In another embodiment the compounds according to formula Ia
are used for the maskless photolithography based synthesis of
carbohydrates, glycoproteins, proteoglycans, and the like, as
OH-protecting group as ether.
[0101] In another embodiment the compounds according to formula Ia
are used as SH-protecting group for orthogonal strategies as ether,
ester or thiocarbonate.
[0102] In another embodiment the compounds according to formula Ia
are used as photoactivable protecting groups for releasing an
biologically active structure for the initiation of a polymerase
reaction or a ATP-dependent biochemical conversion.
[0103] The present disclosure further relates to the use of the
compound according to formula Ia, characterized in that light is
used for the maskless photolithography having a wavelength of 375
to 405 nm. Some embodiments use a wavelength of 390 nm.
[0104] The present disclosure further relates to a method for
producing the diarylsulfide backbone containing PLPG which can be
used for the photolithography based oligonucleotide and peptide
synthesis, wherein the method comprises the following steps: [0105]
a) Providing as a starting material p-Diethylbenzene. [0106] b)
Bromination of the phenyl ring in one position by the action of
molecular bromine and purification by distillation. [0107] c)
Nitration of the obtained compound in Nitric- and Sulfuric Acid in
the position para- to the Bromine and isolation and purification by
column chromatography on silica gel and crystallization. [0108] d)
Hydroxymethylation of the compound by the action of
para-Formaldehyde in DMSO and Triton B at the benzylic position.
[0109] e) Conversion of the aromatic bromine group to the aryl
sulfide by action of the appropriate thiophenol, thionaphthol etc
in DMF potassium carbonate and catalytic amounts of copper(II) salt
and purification by column chromatography on silica gel. [0110] f)
Conversion of the previous alcohol to the chlorocarbonate by action
of triphosgen in THF and triethylamine. [0111] g) Reaction of the
chlorocarbonate with the appropriate nucleoside and further
reacting the nucleoside with a phosphitylating agent to the
appropriate phospshoramidite, or [0112] reaction of the
chlorocarbonate with the appropriate amino acid derivative.
[0113] The following examples are provided to aid the understanding
of the present disclosure, the true scope of which is set forth in
the appended claims. It is understood that modifications can be
made in the procedures set forth without departing from the spirit
of the disclosure.
[0114] Further embodiments are included by the following items:
[0115] 1. A compound of the formula
##STR00010##
[0115] wherein A is selected from the group consisting of
--CH.sub.2--, --CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH(CH.sub.3)--CH.sub.2--, and R1 is an unsubstituted or
substituted aryl- or heteroaryl-group, and R3 is H, a methyl group
or an ethyl group, and wherein R2 is
##STR00011##
or wherein R2 is
##STR00012##
wherein R4 is H, forms a phosphoramidite, H-phosphonate or
phosphate triester, and wherein R5 is H, OH, a halogen or XR6,
wherein X is O or S and R6 is H, an alkyl-group, aryl-group, or OR6
forms a phosphoramidite, phosphodiester, phosphotriester,
H-phosphonate or an acetal or silicone moiety, and wherein B is
selected from the group consisting of adenine, cytosine, guanine,
thymine, uracil, 2,6-diaminopurine-9-yl, hypoxanthin-9-yl,
5-methylcytosinyl-1-yl, 5-amino-4-imidazolecarboxylic acid-1-yl or
5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein when B is
adenine, cytosine or guanine the primary amino group optionally has
a protecting group or when B is thymine or uracil at the O.sup.4
position is optionally a protecting group, or wherein R.sub.2
is
##STR00013##
wherein R7 is a natural amino acid, a non-natural amino acid or an
amino acid derivative forming an urethan bond to formula Ia, or
wherein formula IV represents the carboxy function of a natural
amino acid, a non-natural amino acid or an amino acid derivative,
forming an ester bond to formula Ia. [0116] 2. The compound
according to item 1, characterized in that R1 is a phenyl-group, a
tert-butyl-phenyl group, a 1- or 2-naphthyl-group or a
2-pyridyl-group. [0117] 3. The compound according to item 1 or 2,
characterized in that A is --CH(CH.sub.3)--CH.sub.2--. [0118] 4.
The compound according to items 1 to 3, characterized in that R3 is
H or an ethyl group. [0119] 5. The compound according to items 1 to
4, characterized in that R4 is H and R5 is H. [0120] 6. The
compound according to items 1 to 5, characterized in that B is
selected from the group consisting of adenine, cytosine, guanine,
thymine or uracil. [0121] 7. The compound according to items 1 to
6, characterized in that, when B is adenine, cytosine or guanine
the protecting group is phenoxyacetyl-,
4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or
dimethylformamidino-residues, when B is adenine the protecting
group is benzoyl- or
p-nitro-phenyl-ethoxy-carbonyl-(p-NPPOC)-residues, when B is
guanine the protecting group is isobutyroyl-, p-nitrophenylethyl
(p-NPE) or p-NPEOC-residues and when B is cytosine the protecting
group is benzoyl-, isobutyryl- or p-NPEOC-residues. [0122] 8. The
compound according to items 1 to 4, characterized in that R7 is a
natural amino acid. [0123] 9. Included in this disclosure is the
use of the compound according to items 1 to 8 as photoactivable
protecting group using maskless photolithography. [0124] 10.
Included in this disclosure is the use of the compound according to
item 7 for the maskless photolithography based DNA array synthesis
as intermediate or permanent OH-protecting group in nucleoside
derivatives at the 3'-OH end or the 5'-OH end. [0125] 11. Included
in this disclosure is the use of the compound according to item 8
for the maskless photolithography based peptide array synthesis as
NH-protecting group in amino acids. [0126] 12. Included in this
disclosure is the use of the compound according to item 8 for the
maskless photolithography based peptide array synthesis as
COOH-protecting group in amino acids. [0127] 13. Included in this
disclosure is the use of the compound according to item 8 for the
maskless photolithography based synthesis of carbohydrates as
OH-protecting group. [0128] 14. Included in this disclosure is the
use of the compound according to item 8 for orthogonal protecting
group strategy as SH-protecting group. [0129] 15. Included in this
disclosure is the use of the compound according to items 8 to 13,
characterized in that light is used for the maskless
photolithography having a wavelength of 374 to 405 nm, for example
390 nm. [0130] 16. Also included in this disclosure is a method for
preparing a diarylsulfide backbone containing photolabile
protecting group according to one of the items 1 to 8 comprising
the steps of [0131] a) Provision of p-diethylbenzene as a starting
material [0132] b) Bromination of the phenyl ring [0133] c)
Nitration of the obtained compound in Nitric- and Sulfuric Acid in
the position para- to the Bromine [0134] d) Purification and
crystallization [0135] e) Hydroxymethylation of the compound at the
benzylic position [0136] f) Conversion of the aromatic bromine
group to the arylsulfide using thiophenol [0137] g) Purification
[0138] h) Conversion of the alcohol to chlorocarbonate [0139] i)
Reaction of the chlorocarbonate with a nucleoside and reaction of
the nucleoside with a phosphitylating agent, or [0140] Reaction of
the chlorocarbonate with an amino acid derivative. [0141] 17. Also
included is a method according to item 16, characterized in that R1
is a phenyl-group, a tert-butyl-phenyl group, a 1- or
2-naphthyl-group or a 2-pyridyl-group. [0142] 18. In other
embodiments of the method according to item 16 or 17, characterized
in that A is --CH(CH.sub.3)--CH.sub.2--. [0143] 19. In certain
embodiments of the method according to item 16 to 18, R3 is H or an
ethyl group.
[0144] The present disclosure further relates to diarylsulfide
chromophore containing PLPG which can be used for the
photolithography based oligonucleotide and peptide synthesis having
the structure
##STR00014##
[0145] wherein Y is S or O, and
[0146] A is selected from the group consisting of --CH2-,
--CH.sub.2--CH.sub.2--, --CH(CH.sub.3)--,
--CH(CH.sub.3)--CH.sub.2--, --CH2-CH(Alky,Aryl)- and
--CH(CH3)-CH(Alkyl, Aryl)-
[0147] R1 is an unsubstituted or substituted aryl- or
heteroaryl-group or a condensed aryl- or heteroaryl-group, and R3
is H, a methyl group or an ethyl group, and
[0148] wherein R2 is H, forms a phosphoramidite, H-phosphonate or
phosphate triester, or
[0149] wherein R2 is
##STR00015##
[0150] or wherein R2 is
##STR00016##
[0151] wherein R4 is H, an alkyl-group, aryl-group, or OR4 forms a
phosphoramidite, H-phosphonate or phosphate triester and
[0152] wherein R5 is H, OH, a halogen or XR6, wherein X is O or S
and R6 is an alkyl-group, aryl-group, or OR6 forms a
phosphitamide-group, phosphodiester, phosphotriester or
H-phosphonate or an acetal or a silicone moiety and
[0153] wherein B is selected from the group consisting of adenine,
cytosine, guanine, thymine, uracil, 2,6-diaminopurine-9-yl,
hypoxanthin-9-yl, 5-methylcytosinyl-1-yl,
5-amino-4-imidazolecarboxylic acid-1-yl or
5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein
[0154] when B is adenine, cytosine or guanine the primary amino
group optionally has a protecting group or when B is thymine or
uracil at the O.sup.4 position is optionally a protecting
group,
[0155] or wherein R2 is
##STR00017##
[0156] wherein R7 is a natural amino acid, a non-natural amino acid
or an amino acid derivative, including but not limited to .alpha.-
or .beta.-amino acids, forming an urethan bond to formula Ib,
[0157] or wherein formula IV represents the carboxy function of a
natural amino acid, a non-natural amino acid or an amino acid
derivative, forming an ester bond to formula Ib, including but not
limited to .alpha.- or .beta.-amino acids.
[0158] In another embodiment compounds according to formula Ib are
used, characterized in that R1 is a phenyl-group, a
tert-butyl-phenyl group, a 1- or 2-naphthyl-group, an
aminophenyl-group, an N-alkylaminophenyl-group, an
N-Acylaminophenyl-group, a carboxyphenyl-group, a phenylcarboxylic
ester, an amide or a 2- or 4-pyridyl-group, A is
--CH(CH.sub.3)--CH.sub.2--, R2 is a phosphoramidite or
--P(OCH.sub.2 CH.sub.2 CN)(N-iPr.sub.2), R3 is H or an ethyl group,
R4 is H and R5 is H, R4 is H and R5 is OH or OSi(Alkyl3).
[0159] In another embodiment compounds according to formula Ib are
used, characterized in that B is selected from the group consisting
of adenine, cytosine, guanine, thymine, 5-methylcytosine or
uracil.
[0160] In another embodiment compounds according to formula Ib are
used, characterized in that, when B is adenine, cytosine or guanine
the protecting group is phenoxyacetyl-,
4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or
dimethylformamidino-residues, when B is adenine the protecting
group is a benzoyl-residue, when B is guanine the protecting group
is a isobutyroyl-residue and when B is cytosine the protecting
group is benzoyl- or isobutyroyl-residues.
[0161] In another embodiment compounds according to formula Ib are
used, characterized in that R7 is a natural amino acid.
[0162] The present disclosure further relates to the use of the
compounds according to formula Ib as photoactivable protecting
groups using maskless photolithography. In one embodiment of the
disclosure micro mirror devices are used to perform a spatial
selective exposure of the oligonucleotide and peptide microarrays
to visible light in order to deprotect nucleotides and amino acids,
respectively, in the exposed areas during the synthesis process.
Deprotection of nucleotides and amino acids, respectively, lead to
the release of the next linkage site for the respective next
nucleotide or amino acid. The next nucleotide or amino acid which
should be coupled to the released linkage site within the specific
areas is simply added by its provision within a solvent plus an
activating reagent which is poured onto the array. This strategy is
repeated until oligonucleotides and oligopeptides, respectively, of
the desired lengths and design are obtained. Using this strategy it
is possible to produce highly dense microarrays of at least 10,000
and in certain embodiments 100,000 to 500,000 features per
cm.sup.2.
[0163] Certain embodiments of the PLPG according to the disclosure
can be removed by using visible light in a range from 375 nm to 420
nm, while certain embodiments can be removed in the range from 390
to 405 nm. Certain embodiments are removed via deprotection at
wavelengths of 390 nm and 404 nm, respectively. Both wavelengths
can be generated using light sources which are much less expensive
as compared to light sources necessary to perform deprotection in
the near UV range at approximately 365 nm. In some embodiments,
solid state lasers within the range from 375 nm to 420 nm, or
within the range of 390 nm and 404 nm, are used as light sources to
remove the PLPG according to the disclosure. In some embodiments,
LEDs (light emitting diodes) with sufficient emission within the
range from 375 nm to 420 nm, or within the range of 390 nm and 404
nm, are used as light sources to remove the PLPG according to the
disclosure. Especially LEDs are low cost products as they are
produced in high quantities, e.g. for the use in Blu-ray
Players.
[0164] In a further embodiment micro mirror devices are used, which
are optimized for the use of visible light in the range of 375 nm
to 420 nm, or in the range of 390 to 410 nm, or in the range of 390
nm to 404 nm, respectively. In a further embodiment the coating of
the micro mirror devices remain on the devices in order to be used
with visible light. Devices that are used for UV- or near UV-light
have to be optimized for that purpose, i.e. the coating on the
micro mirror elements has to be removed by polishing.
[0165] In another embodiment, LCD displays or a beam splitter can
be used as virtual masks between the light source and the synthesis
area.
[0166] Photolithographic synthesis of the oligonucleotides and
peptides, respectively, can be performed on a support, for example
a solid support. The support can be made of any material known by
the skilled person used for such a purpose. Examples of materials
useful for the support is are plastic, silicon, diamond carbon or
glass. In some embodiments, plastic or glass is used as a support.
In certain embodiments, as the material is optical grade polyolefin
or optical grade microscope glass slides. The support can be
provided in any form, such as beads, gels, plates, membranes,
slides or chips. The support can be transparent or non-transparent.
In some embodiments, the support exhibits at least 30%, at least
60%, or at least 90% light transmission at a wavelengths of between
375 nm to 410 nm.
[0167] The PLPG according to the disclosure can be used in any
process for oligonucleotide synthesis known by the skilled person
where protected nucleosides or nucleotides are necessary. The
PLPG-nucleotides as described herein can be used for the synthesis
of oligonucleotides in solution, or the PLPG-nucleotides as
described herein can be used for the synthesis of oligonucleotides
on a solid support. The synthesis can be performed by any standard
method known in the state of the art. For example, the synthesis
can be performed by using photolithographic techniques, such as
maskless techniques wherein a micro mirror device is used to expose
light to spatial selected features on a microarray as explained
above.
[0168] Solvents known by the skilled person can be used during
oligonucleotide synthesis, such as acetonitrile.
[0169] The PLPG associated to nucleosides or nucleotides for
oligonucleotide synthesis can be used at various concentrations
within the solvents, such as in the range of 1 mmol/L to 100
mmol/L, 10 mmol/L to 40 mmol/L, or at a concentration of 25
mmol/L.
[0170] The PLPG associated with nucleosides or nucleotides can be
used in connection with sensitizing agents known by the skilled
person, which increase the effectiveness of the deprotection
reaction. Examples of sensitizing agents useful for this disclosure
are benzophenone, xanthone and thioxanthone derivates, like e.g.
thioxanthen-9-one, alkylthioxanthen-9-ones, as for example
isopropylthioxanthen-9-one, 2-ethylthioxanthen-9-one,
2-chloro-thioxanthen-9-one, 1,4-dimethoxythioxanthen-9-one.
[0171] Oligonucleotide microarrays can be used for a variety of
purposes, including but not limited to sequence capturing,
comparative genomic hybridization (CGH), CHIP-chip analysis,
DNA-methylation analysis, gene expression analysis and comparative
genome sequencing.
[0172] In another embodiment compounds according to formula Ib are
used in the maskless photolithography based DNA array synthesis as
intermediate or permanent OH-protecting group in nucleoside
derivatives at the 3'-OH end or the 5'-OH end as carbon ester,
wherein the synthesis can be performed in 3'-5'-direction or in
5'-3'-direction.
[0173] If the PLPG is located at the 5-end, the nucleotide carries
a phosphoramidite group on its 3-end, which can be reacted with a
free --OH group on the solid support to form a stable elongated
oligonucleotide. After all oligonucleotides are synthesized, all
PLPG are removed and the oligonucleotide still bound to the solid
support has a free 5'-OH.
[0174] If however the PLPG is located at the 3'-end, the nucleotide
carries a phosphoramidite group on its 5'-end, which can be reacted
with any free --OH group on the solid support to form a stable
elongated oligonucleotide. After all oligonucleotides are
synthesized, all PLPG are removed and the oligonucleotide still
bound to the solid support has a free 3'-OH.
[0175] The PLPG according to the disclosure can further be used in
any process for peptide synthesis known by the skilled person where
protected amino acids are necessary. The used amino acids can be
non-natural amino acids, amino acid derivatives and/or natural
amino acids PLPG as described herein can be used for the synthesis
of oligopeptides in solution, or the PLPG as described herein can
be used for the synthesis of oligopeptides on a solid support. The
synthesis can be performed by any standard method known in the
state of the art. For example, the synthesis can be performed by
using photolithographic techniques, such as techniques where a
micro mirror device is used to expose visible light to spatial
selected features on a microarray as explained above.
[0176] It has been shown that the deprotection reaction is
dependent on the solvent used during the peptide synthesis process.
Solvents known by the skilled person can be used during peptide
synthesis. For example, polar solvents like dimethylsulfoxide
(DMSO), n-methylpyrrolidone (NMP), acetonitrile (MeCN) or
isopropanol can be used. Said solvents can contain certain
additives, such as imidazole, hydroxylamine and water. Imidazole
can be added at concentrations of 0.1% to 3% (v/v), 0.5% to 1.5%
(v/v), or imidazole can be added at a concentration of 1% (v/v).
Hydroxylamine can be added at concentrations of 0.1% to 3% (v/v),
0.2% to 1% (v/v), or hydroxylamine can be added at a concentration
of 1% (v/v). Water can be added at concentrations of 0.1% to 20%
(v/v), 1% to 17% (v/v), or water can be added at a concentration of
1% (v/v). Examples of useful solvents are DMSO, DMSO+1% imidazole,
NMP+0.5% hydroxylamine, MeCN+1% H.sub.2O, MeCN+1% H.sub.2O+1%
imidazole, isopropanol+1% imidazole, isopropanol+12% H.sub.2O+1%
imidazole.
[0177] The PLPG associated with amino acids for peptide synthesis
can be used in a concentration within the solvents of 0.1 mmol/L to
0.5 mmol/L, 0.2 mmol/L to 0.4 mmol/L, or the PLPG can be used at a
concentration of 0.3 mmol/L.
[0178] The PLPG associated with amino acids can be used in
connection with sensitizing agents known by the skilled person,
which increase the effectiveness of the deprotection reaction.
[0179] Oligopeptide microarrays can be used for a variety of
purposes, including but not limited to screening of antibody
libraries, quantitative or qualitative analysis of biological
samples, biomarker discovery, enrichment of scarce proteins,
depletion of high abundant proteins, analysis of
protein-protein-interactions, analysis of DNA-protein-interactions
or RNA-protein-interactions.
[0180] In another embodiment compounds according to formula Ib are
used for the maskless photolithography based peptide array
synthesis as NH-protecting group in amino acids as urethan. The
PLPG is used as NH-blocked free acid, activated ester, acid
halogenide, anhydride, intermolecular or intramolecular as
N-carboxy-anhydride (NCA).
[0181] In another embodiment the compounds according to formula Ib
are used for the maskless photolithography based peptide array
synthesis as COOH-protecting group in amino acids as ester for
inverse direction of synthesis.
[0182] The PLPG according to the disclosure can further be used in
any process known by the skilled person where protected sugars are
necessary. The sugars used can be compounds, such as aldohexoses
and aldopentoses. In an embodiment, the PLPG as described herein
can be used for the synthesis of carbohydrates, glycoproteins and
proteoglycans in solution. In some embodiments, the PLPG as
described herein can be used for the synthesis of carbohydrates,
glycoproteins and proteoglycans on a solid support. The synthesis
can be performed by any standard method known in the state of the
art. For example, the synthesis can be performed by using
photolithographic techniques, such as techniques where a micro
mirror device is used to expose visible light to spatial selected
features on a microarray as explained above.
[0183] Carbohydrate microarrays can be used for a variety of
purposes, including but not limited to analysis of
saccharide-protein-interactions, high-throughput analysis of
proteins and cells, analysis of glycans and their molecular
interactions,
[0184] In another embodiment the compounds according to formula Ib
are used for the maskless photolithography based synthesis of
carbohydrates, glycoproteins, proteoglycans, and the like, as
OH-protecting group as ether.
[0185] In another embodiment the compounds according to formula Ib
are used as SH-protecting group for orthogonal strategies as ether,
ester or thiocarbonate.
[0186] In another embodiment the compounds according to formula Ib
are used as photoactivable protecting groups for releasing an
biologically active structure for the initiation of a polymerase
reaction or a ATP-dependent biochemical conversion.
[0187] The present disclosure further relates to the use of the
compound according to formula Ib, characterized in that light is
used for the maskless photolithography having a wavelength of 375
to 405 nm, for example 390 nm.
[0188] The present disclosure further relates to a method for
producing the diarylsulfide backbone containing PLPG which can be
used for the photolithography based oligonucleotide and peptide
synthesis, wherein the method comprises the following steps: [0189]
a) Providing as a starting material p-Diethylbenzene. [0190] b)
Bromination of the phenyl ring in one position by the action of
molecular bromine and purification by distillation. [0191] c)
Nitration of the obtained compound in Nitric- and Sulfuric Acid in
the position para- to the Bromine and isolation and purification by
column chromatography on silica gel and crystallization. [0192] d)
Hydroxymethylation of the compound by the action of
para-Formaldehyde in DMSO and Triton B at the benzylic position.
[0193] e) Conversion of the aromatic bromine group to the aryl
sulfide by action of the appropriate thiophenol, thionaphthol etc
in DMF potassium carbonate and catalytic amounts of copper(II) salt
and purification by column chromatography on silica gel. [0194] f)
Conversion of the previous alcohol to the chlorocarbonate by action
of triphosgen in THF and triethylamine. [0195] g) Reaction of the
chlorocarbonate with the appropriate nucleoside and further
reacting the nucleoside with a phosphitylating agent to the
appropriate phospshoramidite, or [0196] reaction of the
chlorocarbonate with the appropriate amino acid derivative.
[0197] The following examples, including sequences and figures, are
provided for the purpose of demonstrating various embodiments of
the instant disclosure and aiding in an understanding of the
present disclosure, the true scope of which is set forth in the
appended claims. These examples are not intended to, and should not
be understood as, limiting the scope or spirit of the instant
disclosure in any way. It is understood that modifications can be
made in the procedures set forth without departing from the spirit
of the disclosure.
EXAMPLES
Example 1
[0198] Evaluation of the Half-life of 5PyS4EtNPPOC-Thymidine in
Dependence of the Solvent at a Wavelengths of 390 nm and 404 nm
[0199] To evaluate the half-life of the PLPG,
5PyS4EtNPPOC-Thymidine was dissolved in a concentration of c=0.3
mmol/L in the solvents given in FIGS. 1 and 2. As solvents
dimethylsulfoxide (DMSO), n-methylpyrrolidone (NMP), acetonitrile
(MeCN) and isopropanol were used. Imidazole, hydroxylamine or water
were added to the solvents as depicted in the table. In case of an
irradiation wavelength of 390 nm (FIG. 1), light exposure was
performed for 2, 4, 6 s or 2, 4, 6, 8 s or 2, 4, 6, 8, 12 s,
respectively, to induce deprotection of threonine. In case of an
irradiation wavelength of 404 nm (FIG. 2), light exposure was
performed for 1, 2, 3, 4 min or 1, 2, 3, 5 min, respectively, to
induce deprotection of thymidine. Subsequently, the solution was
analyzed by HPLC in order to evaluate the time period necessary to
deprotect 50% of the initial amount of the protected thymidine. The
half-lives were then extrapolated from the durations resulting from
the exposure times. As can be taken from FIGS. 1 and 2, in case of
a wavelengths of 390 nm, fastest deprotection was achieved with
DMSO (2.1 s), NMP+0.5% hydroxylamine (1.8 s), MeCN+1% H.sub.2O (2.0
s) and isopropanol+12% H.sub.2O+1% imidazole (2.2 s). In case of a
wavelengths of 404 nm, fastest deprotection was achieved with
DMSO+1% imidazole (2.5 min), NMP+0.5% hydroxylamine (1.8 min),
MeCN+1% H.sub.2O+1% imidazole (2.6 min) and isopropanol+12%
H.sub.2O+1% imidazole (3.8 min). Concerning the significant time
differences between 390 nm (seconds) and 404 nm (minutes), it has
to be taken into consideration that in case of the former the power
output of the lamp was 15 W, whereas the in case of the latter the
power output of the lamp was 0.08 W.
Example 2
[0200] UV Absorption Characteristics of PLPG
[0201] UV absorption for different PLPG at the wavelengths commonly
used is depicted in FIG. 3. The appropriate derivatives of
phenylalanine with the PLPG according to the disclosure were
dissolved at a concentration of 1 mg/mL in UV grade methanol. UV
spectra were recorded in a scanning photometer and absorption
values were taken at the given wavelengths. Molar extinction
coefficients were calculated from the molecular weight using
Lambert Beers law. Deprotection speed of any PLPG is approximately
the product of triplett quantum yield times molar extinction
coefficient. It may thus be estimated, that PhS-phenylalanine
deprotects 15 times more efficient as BTA-phenylalanine and about
25 times more efficient as NPPOC-phenylalanine at an irradiation
wavelength 390 nm.
Example 3
[0202] Synthesis of a Peptide Array Containing the Target Sequence
of an Anti-V5 Antibody Using Disulfide-PLPG-Amino Acids
[0203] Target-epitope: (H)G K P I P N P L L G L D S T-(OH)
[0204] Peptide features on the array were synthesized in a pattern
of varying density on a Roche Nimblegen Maskless Array Synthesizer
according to the synthesis scheme in FIG. 8, at a light dose
of:
[0205] Area 1: 2.5 s irradiation at 190 mW/sqcm [365 nm]
[0206] Area 2: 3.5 s irradiation at 190 mW/sqcm [365 nm]
[0207] Area 3: 4.5 s irradiation at 190 mW/sqcm [365 nm]
[0208] Exposure was in NMP/hydroxylamine (1%). The standard
irradiation time for NPPOC amino acids at this lamp intensity would
have been 50 s. All features of the array contained the same V-5
antigen sequence. Coupling was under standard conditions, 30 mM
amino acid, 30 mM activator (HBTU and HOBT) and 60 mM Hunig base in
peptide grade DMF sequentially coupled a Greiner
3D-Amino-functionalized microscope slide. Washing between cycles
and between coupling and irradiation was with NMP. Final
deprotection of the array was achieved by soaking in trifluoro
acetic acid, water, triisopropylsilane 97.5:2:0.5 for 1 h. After
thorough washing with water, the array was incubated with
anti-V5-antibody (labeled with Cy-3 fluorescent dye), obtained from
Sigma in the manufacturers recommended buffer system at 1:10 000
dilution (0.1 .mu.g/mL) overnight at room temperature. After
washing with buffer and drying the array was scanned at the
appropriate filter setting in a Roche Nimblegen MS 200 fluorescent
scanner at 2 .mu.m resolution. Images were analyzed in Nimblescan
and Genepix (Molecular Dynamics) software packages.
[0209] The results show excellent signal intensity over the three
doses as shown in FIG. 8 b, indicating complete photodeprotection
at less than 500 mW*s, whereas NPPOC-amino acids would require
about 10.000 mW*s to achieve the same result.
[0210] In addition, the same experiment was conducted on a Roche
Nimblegen Maskless Array Synthesizer according to the synthesis
scheme in FIG. 8a, at a Light Dose of:
[0211] Area 1-30: 1-30 s irradiation at 90 mW/sqcm [390 nm]
[0212] Incubation, staining and washing was done as mentioned
above.
[0213] The results show excellent signal intensity with a maximum
at about 21 s irradiation at 390 nm as shown in FIG. 8 c,
indicating complete photodeprotection at less than 2.000 mW*s,
whereas NPPOC-amino acids are insufficiently deprotected at 390 nm
and do not give signals attributable to the peptides made.
Example 4
[0214] Synthesis of Disulfide-PLPG-Amino Acids
[0215] Synthesis pathways are depicted in the respective figures as
indicated below.
[0216] General formula of phenyl-thio-NPPOC-amino acids
##STR00018##
a) 2-(2-Nitro-4-ethyl-5-thiophenyl-phenyl)propanol ("PhSNPPOH")
TABLE-US-00001 [0217] 1,4-Diethylbezene 1902.6 g 14.18 mol 1 Eq
Bromine 2288 g 14.32 mol 1.01 Eq Iron (powder) 26 g
[0218] The corresponding synthesis pathway is depicted in FIG. 4a.
A few drops of bromine are added to a mixture of 1902.6 g
1,4-diethylbenzene and 26 g of iron powder. The mixture is stirred
at ambient temperature until HBr evolution starts. Then the mixture
is cooled in an ice bath and further 2288 g of bromine are added
under vigorous stirring over a period of approximately 5 h. Then
the ice bath is removed and the mixture is stirred over night at
ambient temperature. The reaction mixture is washed with water,
saturated NaHCO.sub.3 solution and again with water. The crude
product is diluted with toluene, concentrated and distilled in
vacuum (approximately 5 mbar/82-84.degree. C.).
[0219] 2740 g 2,5-diethyl-bromobenzene, a colorless liquid, are
obtained (yield: 90% of theory).
[0220] .sup.1H-NMR (300 MHz, DMSO):
[0221] 7.37 ppm (d, 1H, Ar--H); 7.20 ppm (d, 1H, Ar--H); 7.12 ppm
(dd, 1H, Ar--H); 2.65 ppm (q, 2H, Ar--CH.sub.2--CH.sub.3); 2.56 ppm
(q, 2H, Ar--CH.sub.2--CH.sub.3), 1.20-1.15 ppm (m, 6H,
2.times.CH.sub.3).
b) 2,5-Diethyl-4-nitro-bromobenzene
TABLE-US-00002 [0222] 2,5-Diethyl-bromobenzene 426 g 2 mol
HNO.sub.3 (65%) 202 ml H.sub.2SO.sub.4 conc. 241 ml
[0223] The corresponding synthesis pathway is depicted in FIG. 4b.
Under ice cooling 241 mL of H.sub.2SO.sub.4 conc. are added slowly
to 202 mL of HNO.sub.3 (65%). Under ice cooling and vigorous
stirring this mixture is dropped slowly into 426 g of
2,5-diethyl-bromobenzene (dosing time 2 h). The reaction mixture is
stirred over night at ambient temperature. Then the mixture is
poured on ice, diluted with dichloromethane, washed twice with
water and finally with saturated NaHCO.sub.3-solution. The organic
phase is diluted with toluene, concentrated in vacuum and purified
by fast filtration (silica gel, mobile phase iso-hexane).
[0224] 217 g of a slightly yellow oil are obtained (yield: 42% of
theory).
[0225] .sup.1H-NMR (300 MHz, DMSO):
[0226] 7.74 ppm (s, 2H, Ar--H); 4.72 ppm (t, 1H, OH); 3.54-3.48 ppm
(m, 2H, HO--CH.sub.2), 3.26-3.14 ppm (m, 1H, HO--CH.sub.2--CH);
2.74 ppm (q, 2H, Ar--CH.sub.2--CH.sub.3); 1.25-1.15 ppm (m, 6H,
2.times.CH.sub.3).
c) 2-(2-Nitro-4-ethyl-5-bromophenyl)propan-1-ol ("BrEtNPPOH")
TABLE-US-00003 [0227] 2,5-Diethyl-4-nitro-bromobenzene 1000 g 3.87
mol 1 Eq Paraformaldehyde 418.8 g 4.65 mol 1.2 Eq Triton B (40% in
methanol) 1090 ml DMSO 5.2 l Acetic Acid 400 ml
[0228] The corresponding synthesis pathway is depicted in FIG. 4c.
A mixture of 1000 g 2,5-diethyl-4-nitro-bromobenzene, 418.8 g
paraformaldehyde, 1090 mL triton B (40% in methanol) and 5.2 L DMSO
is heated for 2 h at 80 to 90.degree. C. The heating is switched
off and the mixture is stirred for further 4 h. 400 mL of acetic
acid are added. The mixture is diluted with water to a volume of
approximately 15 L and extracted twice with 2 L of toluene. The
toluene extract is washed twice with 1 L of water and then
concentrated in vacuum. The crude product is purified by
chromatography (silica gel, gradient: iso-hexane to
iso-hexane/EtOAc 30%).
[0229] Yield: 521 g 2-(2-Nitro-4-ethyl-5-bromophenyl)propan-1-ol as
a brown oil (46% of theory) plus 77 g of lesser purity.
[0230] .sup.1H-NMR (300 MHz, DMSO):
[0231] 7.74 ppm (s, 2H, Ar--H); 4.72 ppm (t, 1H, OH); 3.54-3.48 ppm
(m, 2H, HO--CH.sub.2), 3.26-3.14 ppm (m, 1H, HO--CH.sub.2--CH);
2.74 ppm (q, 2H, Ar--CH.sub.2--CH.sub.3); 1.25-1.15 ppm (m, 6H,
2.times.CH.sub.3).
d) 2-(2-Nitro-4-ethyl-5-thiophenylphenyl)propanol (PhSNPPOH)
TABLE-US-00004 [0232] BrEtNPPOH 450 g 1.56 mol 1 Eq Thiophenol 172
g 156 mol 1 Eq K.sub.2CO.sub.3 324 g 2.34 mol 1.5 Eq DMF 2 L
[0233] The corresponding synthesis pathway is depicted in FIG. 4d.
A mixture of the reactants and DMF was stirred at 140-160.degree.
C. for 5 h. After cooling to 110.degree. C., the solvent is removed
by distillation under vacuum. The residue was treated with
approximately 2.5 L water and extracted with approximately 1 L
dichlormethane. The organic phase was washed with dilute NaOH and
water, then evaporated to dryness in vacuo, further distilled with
an azeotropic toluene/ethanol-mixture and purified by column
chromatography on silica gel in 5 to 30% ethylacetate in
hexanes.
[0234] Yield: 352 g clear, yellow oil (71%)
e) 2-(2-Nitro-4-ethyl-5-thiophenylphenyl)propanol chlorocarbonate
("PhSNPPOC-Cl")
TABLE-US-00005 [0235] PhSNPPOH 352 g 1.11 mol 1 Eq Triethylamin
112.2 g 1.11 mol 1 Eq Triphosgen 219.4 g 2.22 mol Phosgen 2 Eq THF
ca. 1.7 L
[0236] The corresponding synthesis pathway is depicted in FIG. 4e.
219.4 g triphosgene was dissolved in 1 L dry THF under stirring for
30 min. Under ice-cooling, a solution of 352 g PhSNPPOH and 112.2 g
NEt.sub.3 in 700 mL dry THF was added slowly over a period of 3 h.
After standing overnight, the icebath was replaced by a water bath
at 40.degree. C. and excess phosgen and about 1 L THF removed in
vacuo. The suspension was filtered, the residue washed with little
THF and filtrates evaporated to dryness in vacuo.
[0237] Yield: 410.3 g yellow crystals (97%)
[0238] The material is pure for further use without
purification.
f) PhSNPPOC-Glycine-OH
TABLE-US-00006 [0239] Glycine 5.81 g 0.0774 mol 1 Eq PhSNPPOC--Cl
29.4 g 0.0774 mol 1 Eq Na.sub.2CO.sub.3 18.1 g 0.1703 mol 2.2 Eq
Water 190 mL THF 150 mL
[0240] The corresponding synthesis pathway is depicted in FIG. 4f.
5.81 g glycine und 18.1 g Na.sub.2CO.sub.3 are dissolved in 190 mL
water and 60 mL THF. The solution is stirred in an ice-bath and is
dropwise treated with a solution of 29.4 g PhSNPPOC-Cl in 90 mL
THF. Stirring is continued for 20 min. THF was evaporated and the
solution adjusted to pH 11. The solution is extracted twice with
approximately 500 mL Hexane/Ethylacetate 1:1, the pH is adjusted to
2.5 with dilute HCl and extracted with approximately 500 mL
ethylacetate. The organic phase is washed with approximately 500 mL
water and evaporated to dryness. The product is purified by column
chromatography on silica gel with methanol in dichlormethane (0 to
3%).
[0241] Yield: 21 g pale yellow amorphous foam (65%)
g) PhSNPPOC-Proline-OH
TABLE-US-00007 [0242] Proline 8.6 g 0.075 mol 1 Eq PhSNPPOC--Cl
28.5 g 0.075 mol 1 Eq Na.sub.2CO.sub.3 17.5 g 0.165 mol 2.2 Eq
Water 1000 ml THF 1200 ml
[0243] The corresponding synthesis pathway is depicted in FIG. 4g.
8.6 g proline and 17.5 g Na.sub.2CO.sub.3 are dissolved in 1000 mL
water and 1000 mL THF. The solution is stirred in an ice-bath and
is dropwise treated with a solution of 28.5 g PhSNPPOC-Cl in 200 mL
THF. Stirring is continued for 20 min. THF was evaporated and the
solution adjusted to pH 11. The solution is extracted twice with
approximately 500 mL ethylacetate, the pH is adjusted to 2.5 with
dilute HCl and extracted with approximately 500 mL ethylacetate.
The organic phase is washed with approximately 500 mL water and
evaporated to dryness. The product is purified by column
chromatography on silica gel with methanol in dichlormethane (0 to
2%).
[0244] Yield: 21.7 g pale yellow amorphous foam (63%)
h) PhSNPPOC-Isoleucine-OH
TABLE-US-00008 [0245] Isoleucine 9.97 g 0.076 mol 1 Eq PhSNPPOC--Cl
28.9 g 0.076 mol 1 Eq Na.sub.2CO.sub.3 26.8 g 0.25 mol 3.3 Eq Water
300 ml THF 300 ml
[0246] The corresponding synthesis pathway is depicted in FIG. 4h.
9.97 g isoleucin and 26.8 g Na.sub.2CO.sub.3 are dissolved in 300
mL water and 200 mL THF. The solution is stirred in an ice-bath and
is dropwise treated with a solution of 28.9 g PhSNPPOC-Cl in 90 mL
THF. Stirring is continued for 20 min. THF was evaporated and the
pH of the solution adjusted to 9.5.
[0247] The solution is extracted twice with approximately 500 mL
hexane/ethylacetate 1:1, the pH is adjusted to 3.2 with dilute HCl
and extracted with approximately 500 mL ethylacetate. The organic
phase is washed with approximately 500 mL water and evaporated to
dryness. The product is purified by column chromatography on silica
gel with methanol in dichlormethane (0 to 2%).
[0248] Yield: 15 g pale yellow oil (42%)
i) PhSNPPOC-AsparticAcid-OH
TABLE-US-00009 [0249] Aspartate 10.5 g 0.0789 mol 1 Eq PhSNPPOC--Cl
30.0 g 0.0789 mol 1 Eq Na.sub.2CO.sub.3 23.0 g 0.22 mol 2.8 Eq
Water 1000 ml THF 1200 ml
[0250] The corresponding synthesis pathway is depicted in FIG. 4i.
10.5 g aspartate and 23 g Na.sub.2CO.sub.3 are dissolved in 1000 mL
water and 1000 mL THF. The solution is stirred in an ice-bath and
is dropwise treated with a solution of 30 g PhSNPPOC-Cl in 200 mL
THF. Stirring is continued for 20 min. THF was evaporated. The
solution is extracted twice with approximately 500 mL
hexane/ethylacetate 1:1, the pH is adjusted to 2 with dilute HCl
and extracted with approximately 500 mL ethylacetate. The organic
phase is washed with approximately 500 mL Water and evaporated to
dryness. The product is purified by column chromatography on silica
gel with methanol in dichlormethane (0 to 2%).
[0251] Yield: 28 g pale yellow amorphous foam (74%)
j) PhSNPPOC-Asparagine-OH
TABLE-US-00010 [0252] Asparagine 12.7 g 0.0848 mol 1 Eq
PhSNPPOC--Cl 32.2 g 0.0848 mol 1 Eq Na.sub.2CO.sub.3 19.8 g 0.1866
mol 2.2 Eq Wasser 1000 ml THF 1200 ml
[0253] The corresponding synthesis pathway is depicted in FIG. 4j.
12.7 g asparagine and 19.8 g Na.sub.2CO.sub.3 are dissolved in 1000
mL water and 1000 mL THF. The solution is stirred in an ice-bath
and is dropwise treated with a solution of 32.2 g PhSNPPOC-Cl in
200 mL THF. Stirring is continued for 20 min. THF was evaporated.
The solution is extracted twice with approximately 500 mL ether,
the pH is adjusted to 2 with dilute HCl and extracted with
approximately 500 mL ethylacetate. The organic phase is washed with
approximately 500 mL water and evaporated to dryness. The product
is purified by crystallization from ethylacetate.
[0254] Yield: 28 g pale yellow crystals (73%)
k) PhSNPPOC-Leucine-OH
TABLE-US-00011 [0255] Leucine 12.1 g 0.092 mol 1 Eq PhSNPPOC--Cl
35.0 g 0.092 mol 1 Eq Na.sub.2CO.sub.3 21.5 g 0.202 mol 2.2 Eq
Water 250 ml THF 250 ml
[0256] The corresponding synthesis pathway is depicted in FIG. 4k.
12.1 g leucine and 21.5 g Na.sub.2CO.sub.3 are dissolved in 250 mL
water and 200 mL THF. The solution is stirred in an ice-bath and is
dropwise treated with a solution of 35 g PhSNPPOC-Cl in 50 mL THF.
Stirring is continued for 20 min. THF was evaporated. The solution
is extracted twice with approximately 300 mL hexane/ethylacetate
1:1, the pH is adjusted to 3 with dilute HCl and extracted with
approximately 500 mL ethylacetate. The organic phase is washed with
approximately 500 mL water and evaporated to dryness. The product
is purified by column chromatography on silica gel with methanol in
dichlormethane (0 to 3%).
[0257] Yield: 40 g yellow oil (91%)
l) PhSNPPOC-C.sub.6-Spacer
TABLE-US-00012 [0258] 6-Amino-hexanoic Acid 3.45 g 0.0263 mol 1 Eq
PhSNPPOC--Cl 10.0 g 0.0263 mol 1 Eq Na.sub.2CO.sub.3 6.1 g 0.0579
mol 2.2 Eq Water 300 ml THF 200 ml
[0259] The corresponding synthesis pathway is depicted in FIG. 4l.
3.45 g 6-Amino-hexanoic Acid and 6.1 g Na.sub.2CO.sub.3 are
dissolved in 300 mL water and 120 mL THF. The solution is stirred
in an ice-bath and is dropwise treated with a solution of 10 g
PhSNPPOC-Cl in 80 mL THF. Stirring is continued for 20 min. THF was
evaporated. The pH was adjusted to 10.5. The solution is extracted
twice with approximately 300 mL ether, the pH is adjusted to 2.3
with dilute HCl and extracted with approximately 500 mL
ethylacetate. The organic phase is washed with approximately 500 mL
water and evaporated to dryness. The product is purified by column
chromatography on silica gel with methanol in dichlormethane (0 to
5% and acetic acid 0.5%).
[0260] Yield: 10.4 g pale yellow oil (83%)
m) PhSNPPOC-Lysine(Boc)-OH
TABLE-US-00013 [0261] Fmoc-Lysine(Boc)--OH 37.0 g 0.079 mol 1 Eq
Piperidine 33.6 g 0.395 mol 5 Eq THF 1400 ml Na.sub.2CO.sub.3 18.4
g 0.174 mol 2.2 Eq PhSNPPOC--Cl 30.0 g 0.079 mol 1 Eq
[0262] 37.0 g Fmoc-lysine(Boc)-OH are dissolved in 400 ml THF and
treated with 33.6 g piperidine for 3 h under stirring with a
mechanical stirrer blade. TLC indicated complete FMOC removal after
that period. Water (approximately 2 L) was added and stirred for
another 30 min. The precipitate was filtered with suction. The
clear filtrate was charged with 18.4 g Na.sub.2CO.sub.3 and
evaporated to dryness. Evaporation was continued until all
piperidine was removed, by repeatedly addition of water and
distillation. The residue was dissolved in approximately 1 L water
and treated with 800 mL THF. The solution is stirred in an ice-bath
and is dropwise treated with a solution of 30 g PhSNPPOC-Cl in 200
mL THF. Stirring is continued for 20 min. THF was evaporated. The
pH is adjusted to 2 with dilute HCl and extracted with
approximately 500 mL ethylacetate. The organic phase is washed with
approximately 500 mL Water and evaporated to dryness. The product
is purified by column chromatography on silica gel with methanol in
dichlormethane (0 to 1%).
[0263] Yield: 28.3 g pale orange amorphous foam (60%)
n) PhSNPPOC-Serine(t-Bu)-OH
TABLE-US-00014 [0264] Fmoc-Serine(t-Bu)--OH 30.3 g 0.079 mol 1 Eq
Piperidine 33.6 g 0.395 mol 5 Eq THF 1600 ml Na.sub.2CO.sub.3 18.4
g 0.174 mol 2.2 Eq PhSNPPOC--Cl 30.0 g 0.079 mol 1 Eq
[0265] 30.3 g Fmoc-Serine(Boc)-OH are dissolved in 600 mL THF and
treated with 33.6 g piperidine for 3 h under stirring with a
mechanical stirrer blade. TLC indicated complete FMOC removal after
that period. Water (approximately 2 L) was added and stirred for
another 60 min. The precipitate was filtered with suction. The
clear filtrate was charged with 18.4 g Na.sub.2CO.sub.3 and
evaporated to dryness. Evaporation was continued until all
piperidine was removed, by repeatedly addition of water and
distillation. The residue was dissolved in approximately 600 mL
water, filtered and treated with 800 mL THF. The solution is
stirred in an ice-bath and is dropwise treated with a solution of
30 g PhSNPPOC-Cl in 200 ml THF. Stirring is continued for 20 min.
THF was evaporated. The pH is adjusted to 2 with dilute HCl and
extracted with approximately 500 mL ethylacetate. The organic phase
is washed with approximately 500 mL water and evaporated to
dryness. The product is purified by column chromatography on silica
gel with 1% methanol in dichlormethane.
[0266] Yield: 30.9 g pale yellow amorphous foam (77%)
o) PhSNPPOC-Threonine(t-Bu)-OH
TABLE-US-00015 [0267] Fmoc-Thr(t-Bu)--OH 31.4 g 0.079 mol 1 Eq
Piperidin 33.6 g 0.395 mol 5 Eq THF 1300 ml Na.sub.2CO.sub.3 18.4 g
0.174 mol 2.2 Eq PhSNPPOC--Cl 30.0 g 0.079 mol 1 Eq
[0268] 31.4 g Fmoc-Thr(t-Bu)-OH are dissolved in 600 mL THF and
treated with 33.6 g piperidine for 4 h under stirring with a
mechanical stirrer blade. TLC indicated complete FMOC removal after
that period. Water (approximately 3 L) was added and stirred for
another 30 min. The precipitate was filtered with suction. The
clear filtrate was charged with 18.4 g Na.sub.2CO.sub.3 and
evaporated to dryness. Evaporation was continued until all
piperidine was removed, by repeatedly addition of water and
distillation. The residue was dissolved in approximately 600 mL
Water, filtered and treated with 600 mL THF. The solution is
stirred in an ice-bath and is dropwise treated with a solution of
30 g PhSNPPOC-Cl in 100 mL THF. Stirring is continued for 20 min.
THF was evaporated. The pH is adjusted to 2 with dilute HCl and
extracted with approximately 500 mL ethylacetate. The organic phase
is washed with approximately 500 ml water and evaporated to
dryness. The product is purified by column chromatography on silica
gel with 1% methanol in dichlormethane.
[0269] Yield: 30.9 g pale yellow amorphous foam (69%)
p) PhSNPPOC-Histidine(Trt)-OH
[0270] Step 1:
TABLE-US-00016 Fmoc-His(Trt)-OH 100 g 0.161 mol 1 Eq Piperidine 140
g 1.614 mol 10 Eq THF 2000 ml
[0271] 100 g Fmoc-His(Trt)-OH are dissolved in 2000 mL THF and
treated with 140 g piperidine for 2 h under stirring with a
mechanical stirrer blade. TLC indicated complete FMOC removal after
that period.
[0272] Water (approximately 4 L) was added and stirred for another
30 minutes. The precipitate was filtered with suction. The clear
filtrate was concentrated to remove all THF. The pH was adjusted to
2.5 with dilute HCl and the mixture stirred overnight.
[0273] Filtration yielded 53 g colorless crystals (83%), which were
dried in the air overnight.
[0274] Step 2:
TABLE-US-00017 H-His(Trt)-OH 20.9 g 0.0526 mol 1 Eq PhSNPPOC--Cl
20.0 g 0.0526 mol 1 Eq Na.sub.2CO.sub.3 12.3 g 0.116 mol 2.2 Eq THF
800 ml
[0275] The crystals from above, 20 g, and 12.3 g Na.sub.2CO.sub.3
were dissolved in approximately 800 mL water and 700 mL THF. The
solution is stirred in an ice-bath and is dropwise treated with a
solution of 20 g PhSNPPOC-Cl in 100 mL THF. Stirring is continued
for 20 min. THF was evaporated. The pH is adjusted to 1.5 with
dilute HCl and extracted with approximately 500 mL ethylacetate.
The organic phase is washed with approximately 500 mL water and
evaporated to dryness. The product is purified by column
chromatography on silica gel with 0-1% methanol in dichlormethane
and acetic acid (0.01%).
[0276] Yield: 10 g pale amorphous foam (26%)
Example 5
[0277] Synthesis of Disulfide-PLPG-Nucleotides
5'-PhSNPPOC-dB-3'-PA's
a) 5'-PhSNPPOC-dT
TABLE-US-00018 [0278] Thymidine 19.1 g 0.0789 mol 1 Eq PhSNPPOC--Cl
30.0 g 0.0789 mol 1 Eq Pyridine 300 ml Dichloromethane 50 ml
[0279] A solution of 19.1 g thymidine in 300 mL dry pyridine is
stirred in an ice-bath. Dropwise, a solution of 30.0 g PhSNPPOC-Cl
in 50 mL dichloromethane is added.
[0280] After 10 min continued stirring, the solution is washed
twice with 800 mL water and evaporated to dryness. The residue is
co-evaporated with a mixture of toluene/ethanol. Purification was
accomplished by column chromatography on silica gel in methanol (0
to 2.5%) in dichloromethane.
[0281] Yield: 28 g pale yellowish amorphous foam (60%)
b) 5'-PhSNPPOC-dT-3'-PA
TABLE-US-00019 [0282] 5'-PhSNPPOC-dT 27.1 g 0.0463 mol 1 Eq DCI 2.7
g 0.0232 mol 0.5 Eq P-Reagent 13.5 g 0.0449 mol 0.97 Eq
Dichloromethane 300 ml
[0283] A mixture of the reactants, vigorously dried and under
exclusion of moisture, was stirred overnight at room temperature.
Hexane was added until a slight turbidity remains. After 10 min
stirring, the precipitate is filtered by suction and the crude
product purified by column chromatography on silica gel with a
gradient from 65% to 80% ethylacetate in hexane.
[0284] Yield: 29.7 g pale yellowish amorphous foam (81%)
[0285] P-NMR: 144.4 (m) ppm, 94% pure
c) 5'-PhSNPPOC-dC.sup.Ac
TABLE-US-00020 [0286] dC.sup.Ac 28.3 g 0.105 mol 1 Eq PhSNPPOC--Cl
40 g 0.105 mol 1 Eq Pyridin 650 ml Dichlormethan 100 ml
[0287] A solution of 28.3 g N-(acetyl)-2'-deoxy-cytidine was
co-evaporated twice with 200 mL pyridine, dissolved in 250 mL dry
pyridine and stirred in an ice-bath. Dropwise, a solution of 40.0 g
PhSNPPOC-Cl in 100 mL dichloromethane is added. After 10 min
continued stirring, the solution is washed twice with 800 mL water
and evaporated to dryness. The residue is co-evaporated with a
mixture of toluene/ethanol. Purification was accomplished by column
chromatography on silica gel in methanol (0 to 2.5%) in
dichloromethane.
[0288] Yield: 31 g pale yellowish amorphous foam (48%)
d) 5'-PhSNPPOC-dC.sup.Ac-3'-PA
TABLE-US-00021 [0289] 5'-PhSNPPOC-dC.sup.Ac 29.0 g 0.0473 mol 1 Eq
DCI 2.8 g 0.0237 mol 0.5 Eq P-Reagent 13.8 g 0.0459 mol 0.97 Eq
Dichloromethane 300 ml
[0290] A mixture of the reactants, vigorously dried and under
exclusion of moisture, was stirred overnight at room temperature.
Hexane was added until a slight turbidity remains. The crude
product is purified by column chromatography on silica gel with a
gradient from 65% to 80% ethylacetate in hexane.
[0291] Yield: 21.5 g pale yellowish amorphous foam (56%)
[0292] P-NMR: 144.6 (m) ppm, 99% pure
e) 5'-PhSNPPOC-dA.sup.tac
TABLE-US-00022 [0293] dA.sup.tac 46.3 g 0.105 mol 1 Eq PhSNPPOC--Cl
40 g 0.105 mol 1 Eq Pyridine 650 ml Dichloromethane 100 ml
[0294] A solution of 46.3 g
N-(tert-butyl-phenoxyacetyl)-2'-deoxy-adenosine was co-evaporated
twice with 200 mL pyridine, dissolved in 250 mL dry pyridine and
stirred in an ice-bath. Dropwise, a solution of 40.0 g PhSNPPOC-Cl
in 100 mL dichloromethane is added. After 10 min continued
stirring, the solution is washed twice with 800 mL sodium
bicarbonate solution and water and evaporated to dryness. The
residue is co-evaporated with a mixture of toluene/ethanol.
Purification was accomplished by column chromatography on silica
gel in methanol (0 to 1.5%) in dichloromethane.
[0295] Yield: 35 g pale yellowish amorphous foam (42%)
f) 5'-PhSNPPOC-dA.sup.tac-3'-PA
TABLE-US-00023 [0296] 5'-PhSNPPOC-dA.sup.tac 32.3 g 0.0412 mol 1 Eq
DCI 2.4 g 0.0206 mol 0.5 Eq P-Reagent 12.0 g 0.0399 mol 0.97 Eq
Dichloromethane 300 ml
[0297] A mixture of the reactants, vigorously dried and under
exclusion of moisture, was stirred overnight at room temperature.
Hexane was added until a slight turbidity remains. After 10 min
stirring, the precipitate is filtered by suction and the crude
product purified by column chromatography on silica gel with a
gradient from 50% to 65% ethylacetate in hexane.
[0298] Yield: 32 g pale yellowish amorphous foam (79%)
[0299] P-NMR: 144.3 (m) ppm, 99% pure
g) 5'-PhSNPPOC-dG.sup.tac
TABLE-US-00024 [0300] dG.sup.tac 48.2 g 0.105 mol 1 Eq PhSNPPOC--Cl
40 g 0.105 mol 1 Eq Pyridin 800 ml Dichlormethan 60 ml
[0301] A solution of 48.2 g
N-(tert-butyl-phenoxyacetyl)-2'-deoxy-guanosine was co-evaporated
twice with 200 mL pyridine, dissolved in 400 mL dry pyridine and
stirred in an ice-bath. Dropwise, a solution of 40.0 g PhSNPPOC-Cl
in 600 mL dichloromethane is added. After 10 min continued
stirring, the solution is washed twice with 800 mL water and
evaporated to dryness. The residue is co-evaporated with a mixture
of toluene/ethanol. Purification was accomplished by column
chromatography on silica gel in methanol (0 to 5%) in
dichloromethane.
[0302] Yield: 35 g pale yellowish amorphous foam (42%)
h) 5'-PhSNPPOC-dG.sup.tac-3'-PA
TABLE-US-00025 [0303] 5'-PhSNPPOC-dG.sup.tac 34.0 g 0.0425 mol 1 Eq
DCI 2.5 g 0.0212 mol 0.5 Eq P-Reagent 12.4 g 0.0412 mol 0.97 Eq
Dichloromethane 500 mL
[0304] A mixture of the reactants, vigorously dried and under
exclusion of moisture, was stirred overnight at room temperature.
Hexane was added until a slight turbidity remains. The crude
product is purified by column chromatography on silica gel with a
gradient from 50% to 65% ethylacetate in hexane.
[0305] Yield: 24.5 g pale yellowish amorphous foam (58%)
[0306] P-NMR: 144.5 (m) ppm, 98% pure
Example 6
[0307] Synthesis of Further Diarylsulfide PLPG According to the
Disclosure
a) 5-(t-Butylphenyl-thio)-4-ethyl-2-nitrophenyl-2'-propan-1'-ol
(t-Butylthio-NPPOH)
TABLE-US-00026 [0308] 4-t-Bu-Thiophenol 25.0 g 0.150 mol 1.1 Eq
BrEt--NPPOH 39.0 g 0.135 mol 1 Eq K.sub.2CO.sub.3 31.1 g 0.225 mol
1.7 Eq DMF 200 mL
[0309] A mixture of the reactants was stirred at 100.degree. C. for
3 h. DMF was distilled off in vacuo. The residue was dissolved in
dichloromethane, washed twice with water and evaporated to dryness
in vacuo. The obtained residue was suspended in hexanes, stirred
overnight and filtered off by suction. The crystals were dried.
[0310] Yield: 43 g pale yellow powder (85%)
[0311] .sup.1H-NMR (300 MHz, DMSO):
[0312] 7.72 ppm (s, 1H, Nitro-Ar--H); 7.50 ppm (m, 2H, Ar--H
t-Bu-Ph); 7.38 ppm (m, 2H, Ar--H t-Bu-Ph); 6.88 ppm (s, 1H,
Nitro-Ar--H); 4.67 ppm (s, 1H, OH); 3.25-3.21 ppm (m, 3H,
Ar--CH(Me)--CH.sub.2--OH); 2.73 ppm (q, 2H,
Ar--CH.sub.2--CH.sub.3); 1.29 ppm (s, 9H, CH.sub.3 t-Bu); 1.20 ppm
(t, 3H, Ar--CH.sub.2--CH.sub.3; 0.95 ppm (d, 3H,
Ar--CH(CH.sub.3)--CH.sub.2--OH)
b) Naphthyl-thio-NPPOH
TABLE-US-00027 [0313] BrEt--NPPOH 20.6 g 0.0715 mol 1 Eq
2-Thionaphtol 11.5 g 0.0715 mol 1 Eq Kaliumcarbonat 14.8 g 0.107
mol 1.5 Eq DMF 100 mL
[0314] A mixture of the reactants was refluxed for 1.5 h and
further stirred overnight at room temperature. The residue was
diluted with 1.5 L water and extracted with dichloromethane. The
organic extract was washed twice with water, evaporated to dryness
in vacuo and purified by column chromatography on silica gel with
ethylacetate (0-30%) in hexane.
[0315] Yield: 9.0 g yellow oil (34%)
[0316] .sup.1H-NMR (300 MHz, DMSO):
[0317] 8.07 ppm (m, 1H, Naphthyl-H), 8.00-7.90 ppm (m, 3H,
Naphthyl-H); 7.77 ppm (s, 1H, Nitroaromatic-H); 7.61-7.54 ppm (m,
2H, Naphthyl-H); 7.47-7.41 ppm (m, 1H, Naphthyl-H); 7.11 ppm (s,
1H, Nitroaromatic-H); 4.65 ppm (t, 1H, OH); 3.30-3.15 ppm (m, 3H,
Ar--CH(CH.sub.3)--CH.sub.2--OH); 2.77 ppm (q, 2H,
Ar--CH.sub.2--CH.sub.3); 1.21 ppm (t, 3H, Ar--CH.sub.2--CH.sub.3);
0.92 ppm (d, Ar--CH(CH.sub.3)--CH.sub.2--OH)
c) Nitrobenzimidazol-S-NPPOH
TABLE-US-00028 [0318] BrEt--NPPOH 6.0 g 0.0208 mol 1 Eq
2-Merkapto-5-nitro-benzimidazol 4.06 g 0.0208 mol 1 Eq
Kaliumcarbonat 4.3 g 0.0312 mol 1.5 Eq DMF 50 mL
[0319] A mixture of the reactants was refluxed for 3 h and further
stirred overnight at room temperature. The residue was diluted with
0.5 L water and extracted with dichloromethane. The organic extract
was washed twice with water, evaporated to dryness in vacuo and
purified by column chromatography on silica gel with ethylacetate
(0-30%) in hexane.
[0320] Yield: 5.8 g yellow oil (69%)
[0321] .sup.1H-NMR (300 MHz, DMSO):
[0322] 13.35 ppm (s, 1H, N H); 8.35 ppm (dd, 1H,
Nitrobenzimidazole-H); 7.95 ppm (s, 1H, Nitroaromatic-H); 7.75 ppm
(s, 1H, Nitroaromatic-H); 7.63 ppm (1H, d, Nitrobenzimidazole-H);
4.71 ppm (s, 1H, O H); 3.47 ppm (d, 2H, HO--C H.sub.2); 3.19 ppm
(m, 1H, Ar--C H(CH.sub.3)--CH.sub.2--OH); 2.79 ppm (q, 2H, Ar--C
H.sub.2--CH.sub.3), 1.21-1.15 ppm (m, 6H, 2.times.C H.sub.3)
d) Pyridyl-S-NPPOH
TABLE-US-00029 [0323] BrEt--NPPOH 133.5 g 0.463 mol 1 Eq
2-Merkaptopyridin 51.5 g 0.463 mol 1 Eq Kaliumcarbonat 96.0 g 0.695
mol 1.5 Eq DMF 600 mL
[0324] A mixture of the reactants was stirred at 140.degree. C. for
4 h. DMF was distilled off in vacuo. The residue was dissolved in
dichloromethane, washed twice with water and evaporated to an oil
in vacuo. The residue was purified by column chromatography on
silica gel with ethylacetate (0-30%) in hexane.
[0325] Yield: 87.9 g clear yellow oil (60%)
[0326] .sup.1H-NMR (300 MHz, DMSO):
[0327] 8.43 ppm (m, 1H, Py-H); 7.82 ppm (s, 1H, Nitroaromatic-);
7.70 ppm (m, 1H, Py-H); 7.65 ppm (s, 1H, Py-H), 7.21 ppm (m, 1H,
Py-H); 4.75 ppm (t, 1H, O H); 3.47 ppm (t, 2H, HO--C H.sub.2); 3.20
ppm (m, 1H, HO--CH.sub.2--C HCH.sub.3); 2.72 ppm (q, 1H, C
H.sub.2-Benzylic); 1.15 (m, 6H, 2.times.CH.sub.3)
e) 2,5-Diethyl-4-phenoxy-nitrobenzene
TABLE-US-00030 [0328] Phenole 8.0 g 0.085 mol 1.1 Eq NaH (60% in
Parafine) 3.4 g 0.085 mol 1.1 Eq 4-Bromo-2,5-diethyl-nitrobenze
20.0 g 0.078 mol 1 Eq DMF 60 ml
[0329] Under vigorous stirring 3.4 g of NaH (60% in parafine) was
carefully added to a solution of 8.0 g phenole in 60 ml of DMF.
When the gas evolution was finished, 20.0 g of
4-bromo-2,5-diethyl-nitrobenzene were added to the mixture. The
reaction mixture was stirred for 1.5 h at 170.degree. C. Then the
reaction mixture was cooled to ambient temperature and poured into
600 ml of water. The resulting emulsion was extracted with hexane.
The hexane was distilled of and the distillation residue was dried
over night in vacuum. The distillation residue was dissolved in
hexane and purified by column chromatography (Silica/hexane).
[0330] Yield: 2.2 g slightly coloured oil
[0331] .sup.1H-NMR (DMSO):
[0332] 7.96 ppm (s, 1H, Ar--H); 7.48-7.40 ppm (m, 2H, Ph-H);
7.25-7.18 ppm (m, 1H, Ph-H); 7.09-7.03 ppm (m, 2H, Ph-H); 6.78 ppm
(s, 1H, Ar--H); 2.72 ppm (q, 2H, Ar--C H.sub.2--CH.sub.3); 2.68 ppm
(q, 2H, Ar--C H.sub.2--CH.sub.3); 1.18 ppm (t, 3H, Ar--C
H.sub.2--CH.sub.3); 1.07 ppm (t, 3H, Ar--CH.sub.2--C H.sub.3).
Example 7
Alternative Synthesis of the Diarylsulfide-PLPG with R3=H [Formula
I]
a) 3-Acetamido-ethylbenzol
TABLE-US-00031 [0333] 3-Ethyl-anilin 550 g 4.54 mol Acetanhydride
1100 mL
[0334] Within approximately 4 h, 550 g of 3-Ethyl-anilin were added
to acetanhydride. The mixture was stirred overnight at room
temperature (DC-control hexan/EtOAc 1:1). The reaction mixture was
evaporated to dryness in vacuo. The distillation residue was
distilled in high vacuum (Temp.: 210.degree. C., Head-Temp.:
145.degree. C.).
[0335] Yield: 710 g yellow oil (96%).
b) 3-Acetamido-6-nitro-ethylbenzol
TABLE-US-00032 [0336] 3-Acetamido-ethylbenzol 237.0 g 1.452 mol
H.sub.2SO.sub.4 conc. 622 mL HNO.sub.3 conc. 91.0 g
[0337] 237.0 g 3-Acetamido-ethylbenzol were added dropwise to 622
mL H.sub.2SO.sub.4 conc. in that the temperature of the mixture did
not exceed 20.degree. C. The mixture was cooled to -30.degree. C.
Subsequently, 91.0 g HNO.sub.3 conc. were added dropwise, in that
the inner temperature of the mixture did not exceed -20.degree. C.
The mixture was thawed to -10.degree. C. and poured into 1800 g of
ice. The aqueous phase was separated and extracted with 2.times.200
mL of ether. The precipitate of 3-acetamido-6-nitro-ethylbenzol was
combined to with the ether extracts and dissolved therein. The
ether solution was washed with 100 mL of water and evaporated.
c) 3-Ethyl-4-nitro-aniliniumbromide
TABLE-US-00033 [0338] 3-Acetamido-6-nitro-ethylbenzol (raw product)
approximately 1.45 mol Hydrobromic acid (48%) 400 mL
[0339] The raw product was suspended in 400 mL hydrobromic acid
(48%) and heated for 0.5 h to boiling
(3-ethyl-4-nitro-aniliniumbromide starts to crystallize, which is
associated with a significant increase of the reaction volume). The
mixture was cooled to room temperature under stirring and
subsequently cooled to 5.degree. C. on ice. The suspension was
removed by suction, resuspended in 200 mL cold hydrobromic acid
(48%) and filtered again, followed by washing on the nutsch filter
with approximately 50 mL cold hydrobromic acid (48%).
[0340] Yield: 450 g humid product
d) 3-Brom-6-nitro-ethylbenzol
TABLE-US-00034 [0341] 3-Ethyl-4-nitro-aniliniumbromide ca. 450 g
ca. 1.45 mol (raw product, humid) Hydrobromic acid (48%) 250 mL
Water 400 mL NaNO.sub.2 107.6 g Water 550 mL
[0342] The humid product of the previous approach was suspended in
a solution of 250 mL hydrobromic acid (48%) in 400 mL of water. A
solution of 107.6 g NaNO.sub.2 was added dropwise to 550 mL water
on ice, in that the temperature of the mixture did not exceed
12.degree. C. The mixture was stirred for 30 min at 0.degree. C.
and filtered.
[0343] Sandmayer-Conversion:
TABLE-US-00035 Diazoniumsalt-solution ca. 1.45 mol Copper powder
84.9 g CuSO.sub.4 .times. 5H.sub.2O 212.3 g Hydrobromic acid (48%)
670 mL
[0344] The diazoniumsalt-solution was added dropwise to a mixture
of 84.9 g copper powder, 212.3 g CuSO.sub.4.times.5H.sub.2O and 670
mL hydrobromic acid (48%) on ice, in that the temperature of the
mixture did not exceed 15.degree. C. The mixture was stirred over
night at room temperature, filtered and the organic phase was
separated. The aqueous phase was extracted with dichloromethane.
The combined organic phases were filtered under usage of a thin
layer of silica gel and then evaporated to dryness in vacuo. 167.8
g of raw product was yielded. The distillation residue was
distilled in high vacuum (Temp.: 155.degree. C., Head-Temp.:
85.degree. C.).
[0345] Yield: 144.3 g yellow oil (43% via 4 steps).
e) 2-(2-Nitro-5-brom-phenyl)propanol
TABLE-US-00036 [0346] 3-Bromo-6-nitro-ethylbenzol 309.6 g 1.346 mol
1 Eq Paraformaldehyd 42.4 g 1.413 mol 1.05 Eq Kalium-tert-Butylat
37.8 g 0.337 mol 0.25 Eq DMSO 900 mL
[0347] To a solution of 309.6 g 3-bromo-6-nitro-ethylbenzol and
42.4 g paraformaldehyde in 300 mL DMSO, 37.8 g
potassium-tert-butylat was added in small portions, in that the
temperature increased to 40-50.degree. C. The mixture was stirred
over night at room temperature. 900 mL toluol was added to the
mixture and washed with 3.times.450 mL aqueous NaOH (10%) and
subsequently with 450 mL water. The organic phase was evaporated to
dryness in vacuo. 319 g of raw product was yielded, which was then
purified using chromatography:
[0348] Column: 700 g silica gel, diameter 8.5 cm, equilibrated with
n-hexan. The raw product was dissolved in 100 mL toluol and loaded
onto the column. Elution was performed using the following
gradient: 2.5 L n-hexan,
TABLE-US-00037 Ethylacetate/n-hexane 1:100 1 L 1:50 1.5 L 1:20 1 L
1:6 2 L 1:5 1.5 L
[0349] Yield: 236.06 g 2-(2-Nitro-5-bromo-phenyl)propanol
(67%).
f) PhSNPPOH without Ethyl-Group
TABLE-US-00038 [0350] Br--NPPOH 5.0 g 0.0192 mol 1 Eq Ph--SH 2.3 g
0.0211 mol 1.1 Eq K.sub.2CO.sub.3 4.5 g 0.0326 mol 1.7 Eq DMF 50
mL
[0351] A mixture of the above listed components were mixed 3 h at
120.degree. C. and then over night at 70.degree. C. The reaction
mixture was evaporated to dryness in vacuo. Dichloromethane was
added to the distillation residue and washed with water, with
diluted sodium hydroxide and again with water. The organic phase
was evaporated and purified using chromatography (Stationary phase:
Silica gel equilibrated with iso-hexane; Gradient: iso-hexane/5%
ethyl acetate to iso-hexane/20% ethyl acetate).
[0352] Yield: 2.7 g red-brown oil (48%).
[0353] All references cited in this specification are herewith
incorporated by reference with respect to their entire disclosure
content and the disclosure content specifically mentioned in this
specification.
[0354] While this disclosure has been described as having an
exemplary design, the present disclosure may be further modified
within the spirit and scope of this disclosure. This application is
therefore intended to cover any variations, uses, or adaptations of
the disclosure using its general principles. Further, this
application is intended to cover such departures from the present
disclosure as come within the known or customary practice in the
art to which this disclosure pertains.
* * * * *