U.S. patent application number 14/859983 was filed with the patent office on 2016-01-07 for chemical preparation of ubiquitin thioesters and modifications thereof.
The applicant listed for this patent is BEN-GURION UNIVERSITY OF THE NEGEV RESEARCH AND DEVELOPMENT AUTHORITY. Invention is credited to Ashraf BRIK, Leslie ERLICH, Mahmood HAJ-YAHYA, Ajish KUMAR, Liat SPASSER.
Application Number | 20160002286 14/859983 |
Document ID | / |
Family ID | 44368228 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160002286 |
Kind Code |
A1 |
BRIK; Ashraf ; et
al. |
January 7, 2016 |
CHEMICAL PREPARATION OF UBIQUITIN THIOESTERS AND MODIFICATIONS
THEREOF
Abstract
The present invention discloses latent thioester functionalities
attached to the C-terminus of a first polypeptide, or a first
fragment thereof having a Cys residue at its N-terminus, and a
process using this functionality for the preparation of polypeptide
thioesters, in particular of ubiquitin thioesters, this process
comprising preparing a polypeptide or a fragment thereof, being
attached to a latent thioester functionality, which can then be
ligated with a second polypeptide fragment, followed by selective
activation of the latent thioester functionality group, to provide
the requested polypeptide thioester. There are also provided the
polypeptides obtained by this method, specific unnatural amino
acids useful to be incorporated within the polypeptide thioesters,
and kits for preparing them.
Inventors: |
BRIK; Ashraf; (Beer-Sheva,
IL) ; HAJ-YAHYA; Mahmood; (Taybe-Meshulash, IL)
; KUMAR; Ajish; (Kochi, IN) ; ERLICH; Leslie;
(Hertzilya, IL) ; SPASSER; Liat; (Hertzilya,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BEN-GURION UNIVERSITY OF THE NEGEV RESEARCH AND DEVELOPMENT
AUTHORITY |
Beer-Sheva |
|
IL |
|
|
Family ID: |
44368228 |
Appl. No.: |
14/859983 |
Filed: |
September 21, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13578014 |
Oct 31, 2012 |
9175053 |
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PCT/IL2011/000138 |
Feb 9, 2011 |
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14859983 |
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61302559 |
Feb 9, 2010 |
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Current U.S.
Class: |
530/324 |
Current CPC
Class: |
C07K 14/00 20130101;
C07K 14/47 20130101; C07K 1/1075 20130101; C07K 14/001 20130101;
C07K 1/1077 20130101 |
International
Class: |
C07K 1/107 20060101
C07K001/107; C07K 14/00 20060101 C07K014/00 |
Claims
1. A process for the preparation of ubiquitin thioesters, said
process comprising: a. Chemically synthesizing a first modified
ubiquitin polypeptide fragment selected from CKIQDKEGIPPDQQRLIF
(Ub28-45) (SEQ ID NO: 10), CGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
(Ub46-76) (SEQ ID NO: 8) and
CKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (UB28-76) (SEQ ID
NO: 9), being attached to an N-S acyl trnsfer device on the
C-terminal of said ubiquitin fragment, further wherein said
ubiquitin fragment contains an unprotected Cysteine amino acid on
the N-terminal side thereof; b. chemically synthesizing a second
ubiquitin fragment being complimentary to said first ubiquitin
fragment obtained in step a, wherein said second ubiquitin fragment
is in a thioester form; c. ligating said first ubiquitin fragment
with said second ubiquitin fragment, to obtain an modified
unprotected ubiquitin polypeptide attached to said N-S acyl
transfer device; d. reacting said modified ubiquitin polypeptide
attached to said N-S acyl transfer device with an external thiol
under acidic conditions to obtain said ubiquitin thioester; wherein
said N-S acyl transfer device has the general Formula I:
##STR00017## wherein R, R.sub.1, R.sub.2 and R.sub.3 are selected
from: i. R=hydrogen or 2-nitrobenzyl; R.sub.1=hydrogen or methyl;
R.sub.2=CONH.sub.2; R.sub.3=hydrogen; ii. R=hydrogen or
2-nitrobenzyl; R.sub.1=hydrogen or methyl; R.sub.2=CO--N-pyrroline;
R.sub.3=hydrogen; iii. R=hydrogen or 2-nitrobenzyl; R.sub.1=methyl,
ethyl or benzyl; R.sub.2=hydrogen; R.sub.3=hydrogen; iv. R=hydrogen
or 2-nitrobenzyl; R.sub.1=C1 alkyl-CONH.sub.2 or C1 alkyl-COOH;
R.sub.2=hydrogen; R.sub.3=hydrogen.
2. The process of claim 1, further comprising desulfurization of
said ubiquitin thioester to turn said Cys amino acid into an Ala
amino acid, either before or after step (d).
3. The process of claim 1, wherein said N-S acyl transfer device is
a residue of N-methyl cysteine.
4. The process of claim 1, wherein said ubiquitin thioester
contains at least one protected .delta.-mercaptolysine.
5. The process of claim 4, wherein said protected
.delta.-mercaptolysine is a thiazolidine (Thz)-protected
mercaptolysine.
6. The process of claim 1, wherein said ubiquitin thioester is
prepared of two ubiquitin fragments by native chemical ligation
(NCL), wherein said fragment attached to said N-S acyl transfer
device is: CGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub46-76) (SEQ ID NO: 8)
and said second fragment being in its thioester form is
LQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF (Ubl-45) (SEQ ID NO:
4).
7. The process of claim 1, wherein said ubiquitin thioester is
prepared of two ubiquitin fragments by native chemical ligation
(NCL), wherein said fragment attached to said N-S acyl transfer
device is: CKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG
(Ub28-76) (SEQ ID NO: 9) and said second fragment in a thioester
form is LQIFVKTLTGKTITLEVEPSDTIENVK (Ub1-27) (SEQ ID NO: 6).
8. The process of claim 1, wherein said ubiquitin polypeptide is
prepared of three ubiquitin fragments by native chemical ligation
(NCL), wherein said first fragment attached to said N-S acyl
transfer device is CGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub46-76) (SEQ
ID NO: 8), said second fragment attached to said LTF is
CKIQDKEGIPPDQQRLIF (Ub28- 45) (SEQ ID NO: 10) and said third
fragment in its thioester form is LQIFVKTLTGKTITLEVEPSDTIENVK
(Ub1-27) (SEQ ID NO: 6).
9. The process of claim 1 wherein in at least one of said
fragments, the amino acid K stands for a modified Lysine amino
acid.
10. The process of claim 9, wherein said modified Lysine amino acid
is a protected .delta.-mercaptolysine.
11. The process of claim 10, wherein said protected
.delta.-mercaptolysine is thiazolidine (Thz)-protected
mercaptolysine.
Description
[0001] The topic of peptide thioesters synthesis has captured the
interest of many research groups motivated by the importance of
thioester peptides in native chemical ligation and in protein
synthesis.
[0002] Current methods to prepare peptide thioesters employ Boc- or
Fmoc-Solid phase peptide synthesis (SPPS) approach in combination
with methods relying on the use of safety catch linkers, N-acyl
urea based chemistry and N-S acyl transfer devices employing latent
thioester functionalities. One example of a N-S acyl transfer
method combined with Boc- or Fmoc-SPPS is the reaction of
N-alkylated Cys at the C-terminal peptide with 3-mercaptopropionic
acid (MPA) to generate the desired peptide thioester (for example,
F. Nagaike et al., Org. Lett., 2006, 8, 4465).
[0003] However, these methods are good for peptide fragments up to
30-40 amino acids and are difficult, and sometimes even impossible,
to apply for peptides having more than 70 amino acids.
[0004] Ubiquitin (Ub) is a highly conserved globular 76-residue
eukaryotic protein found in the cytoplasm and nucleus of cells.
Ubiquitin exists both as a monomer and as isopeptide-linked
polymers known as poly-ubiquitin chains.
[0005] The in-vivo process of ubiquitylation serves as a
recognition marker for degradation (in the case of
polyubiquitylation) and to regulate different biochemical processes
(in monoubiquitylation). Three distinct enzymes, known as the E1-E3
system, collaborate to achieve a site-specific ubiquitylation of
the lysine residue (s) in the target protein. The activation of
.alpha.-COOH of ubiquitin is achieved in an ATP dependent manner
using the E1 enzyme, which forms a thioester with the carboxyl
group of Gly76. This step activates ubiquitin and triggers a
nucleophilic attack by the conjugating enzyme E2. The latter
transiently carries the activated ubiquitin, also as a thioester
intermediate, and with the assistance of the E3 ligase transfers
ubiquitin to a specific lysine residue of the protein
substrate.
[0006] It can be seen that ubiquitin thioesters (UbSRs) are key
intermediates in the ubiquitylation of proteins, and it is of great
interest to be able to synthesize & modify them and study their
properties.
[0007] However, since ubiquitin is made of 76 amino acids, applying
the Boc- or Fmoc-SPPS-based methods described above to prepare
thioester derivatives thereof, is very challenging and would
require alternative means to achieve such a goal.
[0008] Indeed, the current methods to prepare Ub-SRs are relying on
either the use of the enzymatic machinery E1-E2 or on expressed
protein technology. However, these approaches are limited mainly to
natural amino acids modification, thereby inhibiting chemical
manipulation of ubiquitin. The chemical synthesis of protein
thioester, wherein unnatural amino acids could be incorporated into
the sequence, remains a synthetic challenge.
[0009] Kent and coworkers have applied native chemical ligation
(NCL) for the synthetic preparation of peptides in general (U.S.
Pat. No. 7,030,217) and Ub peptides in particular (Bang et al,
Angew. Chem. Int. Ed. Eng., 44, 3852-3856, 2005) and have later
reported a "kinetically controlled ligation" strategy for preparing
protein thioesters (for example, D. Bang et al., Angew. Chem. Int.
Ed., 2006, 45, 3985). This approach exploits the different
reactivities of aryl and alkyl thioesters as well as the
differences in the bulkiness of the C-terminal residue of the
thioester peptides for a convergent protein synthesis. However, the
process disclosed by Kent et al. could lead to an undesirable
outcome when the C-terminal residue of the thioester peptides is
intrinsically reactive in peptide ligation, as is the case in
ubiquitin where the C-terminal residue is Gly.
[0010] Therefore, there is a continuing need to develop new
processes for the chemical synthesis of Ub-SRs and analogs
thereof.
[0011] The inventors have now developed a thioester "switchable
device", also termed latent thioester functionality, that is
attached to a solid support and can then be the first building
block in "growing" or elongating a peptide, such as ubiquitin, on
the solid support to obtain the desired protein. This can be done
either in one step or by the use of native chemical ligation of
smaller fragments of this peptide, as long as the C-terminal
fragment of the peptide shall be the one attached to this thioester
"switchable device". Once the required protein has been obtained,
the "switchable device" can be turned into a thioester by reacting
it with an external thiol under acidic conditions, preferably at a
pH lower than 4, most preferably at a pH of about 2.
[0012] This is an important advantage of the latent thioester
functionality, since in contrast to common linkers used in NCL, it
will not be removed neither upon removal from the solid support,
nor under ligation conditions, which are at a pH of about 7 and
higher, and needs an activating step of lowering the pH to below 4
for it to be removed. In other words, the LTF group can be
"switched off" at a specific point in time, by lowering the pH, as
detailed herein.
[0013] Thus, according to one aspect of the invention there is
provided a process of chemically preparing polypeptide thioesters,
this process comprising: [0014] a) Attaching a Latent Thioester
Functionality (LTF) to a solid support; [0015] b) Chemically
synthesizing a polypeptide or a fragment thereof on this solid
support, followed by removal from the solid support, to obtain an
N-terminal unprotected polypeptide or an N-terminal unprotected
fragment thereof, attached to a latent Thioester Functionality on
its C-terminal; [0016] c) If a polypeptide fragment is obtained in
step b: [0017] i. Chemically synthesizing in one or more steps, a
complimentary fragment to the fragment obtained in step b, whereas
the complimentary fragment is prepared as a thioester; [0018] ii.
Reacting the thioester of the complimentary fragment with the
fragment attached to the latent Thioester Functionality obtained in
step b, by native chemical ligation (NCL) to obtain a polypeptide
attached to the latent Thioester Functionality; [0019] d) Reacting
the polypeptide being attached to the latent Thioester
Functionality with an external thiol under acidic conditions to
obtain the polypeptide thioester.
[0020] The term "polypeptide" as used herein refers to a sequential
chain of amino acids linked together via peptide bonds and
encompasses an amino acid chain of any length. If a single
polypeptide can function as a unit, the terms "polypeptide" and
"protein" may be used interchangeably, however, in general, the
term does include peptides, proteins, fusion proteins,
oligopeptides, cyclic peptides, and polypeptide derivatives.
[0021] The term "polypeptide fragment" is used interchangeably with
the term "polypeptide segment" and refers to a peptide or
polypeptide, having either a completely native amide backbone or an
unnatural backbone or a mixture thereof, ranging in size from 2 to
1000 amino acid residues, preferably from 2-99 amino acid residues,
more preferably from 10-60 amino acid residues, and most preferably
from 20-40 amino acid residues. Each peptide fragment can comprise
native amide bonds or any of the known unnatural peptide backbones
or a mixture thereof. Each peptide fragment can be prepared by any
known synthetic methods, including solution synthesis, stepwise
solid phase synthesis, segment condensation, and convergent
condensation.
[0022] The term "unnatural amino acid" refers to any amino acid,
modified amino acid, and/or amino acid analogue, that is not one of
the 20 common naturally occurring amino acids or the rare naturally
occurring amino acids e.g., selenocysteine or pyrrolysine.
[0023] As used herein, the term "thioester", interchangeably used
with the term "thioloester", refers to a moiety represented by
--COSR, often connected to a peptide.
[0024] The term "peptide thioester" or "polypeptide in its
thioester form" may be represented as "peptide-.alpha.-COSR". The R
group in this case may be any number of groups, including 1-15 C
functionalized alkyl, straight or branched, 1-15 C aromatic
structures, 1-4 amino acids or derivatives thereof, preferably
wherein the R group is selected such that the peptide-.alpha.-COSR
is an activated thioester.
[0025] The term "Chemically synthesizing" refers to the fact that
the obtaining of the polypeptide, and in particular obtaining the
polypeptide thioester, is not conducted enzymatically or by gene
expression, neither in vivo nor in vitro.
[0026] The term "Latent thioester Functionality", used
interchangeably with the term "thioester device" or "switchable
device", describes any functionality that is able to undergo a
S-->N acyl transfer and withstand the removal from the solid
support, as well as the ligation conditions. This functionality
therefore serves to introduce into the polypeptide structure, a
precursor to the thioester group to be unmasked at the last stages
of the reaction, only upon an activation step, upon providing
acidic conditions.
[0027] Preferably, the "Latent thioester Functionality" has the
general structure outlined in Formula I:
##STR00001##
[0028] Wherein:
[0029] R is either hydrogen or a thiol protecting group;
[0030] The compound of formula I would attach to the growing
peptide through the Nitrogen attached to R.sub.1, whereas:
[0031] R.sub.1 is selected from the group consisting of: hydrogen,
C1-C3 alkyl, C1-C3 alkyl-COOH, C1-C3 alkyl-CONH.sub.2, C1-C3
alkylene-CONH.sub.2, C1-C3 alkylene-CO.sub.2H, SO.sub.2-alkyl;
SO.sub.2-alkyl-CONH.sub.2, benzyl and derivatives thereof,
alkyl-nitrile and alkyl-halogens. Additional or specific examples
include: iodomethyl, nitromethyl, derivatives of benzyl like
o-nitro-benzyl, p-nitro benzyl;
[0032] Preferably, R.sub.1 is selected from hydrogen, C1-C3 alkyl,
C1-C3 alkyl-CONH.sub.2, SO.sub.2-C1-C3 alkyl-CONH.sub.2, C1-C3
alkyl-COOH.
[0033] More preferably, R.sub.1 is selected from hydrogen, methyl,
ethyl, C1-CONH.sub.2 and C1-COOH.
[0034] R.sub.2 and R.sub.3 are selected from the group consisting
of: hydrogen, CO.sub.2H, CH.sub.2CO.sub.2H, --CH.sub.2OH,
CONH.sub.2, CH.sub.2--CONH.sub.2 and CH.sub.2NH.sub.2, as well as
N-protected derivatives thereof.
[0035] Preferably, R.sub.2 is selected from hydrogen, CONH.sub.2
and N-protected derivatives thereof. This includes for example
CO--N(prolyne amino acid).
[0036] According to one specific embodiment of the invention,
R.sub.3 is hydrogen.
[0037] Furthermore, at least one of R.sub.1 and R.sub.2 should
contain a linking group CONH.sub.2 or N-protected derivatives
thereof, that would be attached to the solid support.
[0038] As noted herein, optionally, the thiol side chain (R) in
this latent thioester functionality is protected to avoid
intramolecular N--S acyl transfer in the cleavage step from the
SPPS resin.
[0039] Examples of thiol-protecting groups include, but are not
limited to, triphenylmethyl (trityl, Trt), acetamidomethyl (Acm),
benzamidomethyl, 1-ethoxyethyl, acetyl, benzoyl, substituted and
unsubstituted benzyl groups and the like.
[0040] Preferably, the thiol-protecting group is a substituted
benzyl group, whereas the phenyl group is substituted by an alkoxy,
such as methoxy, ethoxy and the like or by a nitro group.
[0041] Most preferably, the thiol protecting group is a
photo-labile thiol group, such as 2-nitrobenzyl.
[0042] Upon completion of the ligation reaction, the
thiol-protecting group, if present, is removed (for example by UV),
followed by treatment of the fully unprotected polypeptide with a
thiol, such as MPA, under acidic conditions (i.e. pH<4), to
afford the target polypeptide-thioester.
[0043] According to some preferred embodiments of the invention,
exemplified below, the Latent Thioester Functionality (LTF) is
selected from: [0044] i. R=hydrogen or 2-nitrobenzyl;
R.sub.1=hydrogen or methyl; R.sub.2=CONH.sub.2; R.sub.3=hydrogen;
[0045] ii. R=hydrogen or 2-nitrobenzyl; R.sub.1=hydrogen or methyl;
R.sub.2=CO--N-pyrroline; R.sub.3=hydrogen; [0046] iii. R=hydrogen
or 2-nitrobenzyl; R.sub.1=methyl, ethyl or benzyl;
R.sub.2=hydrogen; R.sub.3=hydrogen; [0047] iv. R=hydrogen or
2-nitrobenzyl; R.sub.1=C1 alkyl-CONH.sub.2 or C1 alkyl-COOH;
R.sub.2=hydrogen; R.sub.3=hydrogen.
[0048] Most preferably, the Latent Thioester Functionality (LTF) is
N-methyl cysteine. In this case, R.sub.1 is methyl; R.sub.2 is
CONH.sub.2 and R.sub.3 is hydrogen. The inventors have shown that
the N-methyl cysteine reacts as expected, both when R is hydrogen
and both when it is 2-nitrobenzyl.
[0049] Examples of some protected latent thioester functionality
attached to peptides, and their reactions to obtain the
polypeptide-thioesters, are shown in scheme below:
##STR00002##
[0050] Scheme 1B is similar, referring in particular to cases when
the "peptide" in scheme 1A is ubiquitin, and when no protection
exists on the LTF's thiol group (R in formula I being
hydrogen).
##STR00003##
[0051] The term, "solid support" is used interchangeably with the
term "solid Phase" and refers to a material having a surface and
which is substantially insoluble when exposed to organic or aqueous
solutions used for coupling, deprotecting, and cleavage
reactions.
[0052] Examples of solid support materials include glass, polymers
and resins, including polyacrylamide, PEG, polystyrene PEG-A,
PEG-polystyrene, macroporous, POROS.TM., cellulose, reconstituted
cellulose (e.g. Perloza), nitrocellulose, nylon membranes,
controlled-pore glass beads, acrylamide gels, polystyrene,
activated dextran, agarose, polyethylene, functionalized plastics,
glass, silicon, aluminum, steel, iron, copper, nickel and gold.
Such materials may be in the form of a plate, sheet, petri dish,
beads, pellets, disks, or other convenient forms.
[0053] Some of the examples and embodiments described herein refer
to resins, which are a type of solid support, and one of ordinary
skill in the art would understand that such examples are not meant
to be limited to resins, but to solid phases in general.
[0054] As used herein, the phrase "peptide attached to a solid
support via a latent thioester functionality" refers to a solid
phase-bound peptide, comprising at least one peptide fragment bound
to a solid phase via a functionality that is able to undergo a
S-->N acyl transfer and withstand the ligation conditions (pH of
about 7 and higher), be stable to stepwise solid phase chemistries,
be able to be covalently linked in unprotected form to the solid
phase, and be cleavable without damaging the assembled
polypeptide.
[0055] According to a preferred embodiment of the invention, the
latent thioester functionality is attached to the solid support via
an amide bond.
[0056] Most important, in order to obtain the thioester as the
final product, the latent thioester functionality must remain
attached to the polypeptide, or to the fragment thereof, during the
removal of each fragment from the solid support, as well as
throughout ligation and be selectively removed therefrom, only upon
activation, such as providing acidic conditions, and reaction with
an external thiol at the last stages of the reaction.
[0057] In particular, the term "ligation conditions" refers to 6 M
Gn.HCl, 200 mM phosphate buffer, pH of about 7 (from about 7 to
about 8) for a period ranging from 4 hours to 48 hours.
[0058] The term "removal from the solid support" refers to cleavage
of the polypeptide or peptide fragment containing the latent
thioester functionality, from the solid support. It is essential
that during this stage, the entire peptide-LTF is cleaved from the
solid support, and that the LTF remains indeed attached to the
peptide or the fragment thereof.
[0059] The following conditions can be used for cleavage of the
peptide-LTF to release the assembled polypeptide from the solid
phase using TFA/TIS/H.sub.2O (95:2.5:2.5).
[0060] The polypeptide fragment obtained by SPPS is an unprotected
polypeptide or fragment. Namely, it does not contain protection
groups on the side chains of the amino acids.
[0061] In particular, it should be noted that the latent thioester
functionality is attached to the C-terminal fragment of the grown
peptide, and that the N-terminal of the polypeptide remains
unprotected, this being an advantage of Native Chemical
Ligation.
[0062] The term "N-terminal" is interchangeably used with
"N-terminus" or "N-terminus amino acid" and refers to mean, as used
herein, the amino acid whose carboxyl group participates in the
formation of the peptide bond, but which has a free amino group. In
a linear peptide, the N terminus is conventionally written to the
left.
[0063] The term "C-terminal" is interchangeably used with
"N-terminus" or "C-terminus amino acid" and refers to mean, as used
herein, the amino acid whose amino group participates in the
formation of the peptide bond, but which still has a free carboxyl
group. In a linear peptide, the C-terminus is conventionally
written to the right.
[0064] If the polypeptide or peptide attached to the latent
thioester functionality, is a fragment of the complete desired
polypeptide to be made into a thioester form, then a second,
complimentary fragment must be prepared separately from the first
fragment.
[0065] The term "complimentary fragment" as used herein refers to a
peptide fragment that, when attached to the fragment obtained in
step b, forms the complete sequence of the desired polypeptide.
[0066] The complimentary fragment can be made in one or more steps,
as required.
[0067] The preparation of the peptide fragments is preferably
conducted by SPPS, according to techniques known to those skilled
in the art.
[0068] Preferably, the SPPS is an Fmoc synthesis, but Boc synthesis
can also be used.
[0069] The term "Native chemical ligation" as used herein refers to
chemoselective reactions involving ligation of a first unprotected
amino acid, peptide or polypeptide and a second unprotected amino
acid, peptide or polypeptide resulting in the formation of an amide
bond having a backbone structure indistinguishable from that of a
peptide or polypeptide occurring in nature or via recombinant
expression. The Native Chemical Ligation is conducted according to
techniques known to those skilled in the art.
[0070] Although several ligation reactions can be conducted to
obtain the final peptide from fragments comprising it, either on
the main backbone of the polypeptide, or via side chains thereof,
preferably, the ligation reaction is between a Cysteine amino acid
on the C-terminal of the polypeptide and a thioester on the
N-terminal the polypeptide. Therefore, the fragments are preferably
prepared such that the N-terminal the polypeptide would be in a
thioester form, and that the C-terminal of the polypeptide would
contain a Cys terminal amino acid, or an equivalent thereof.
[0071] The cys amino acid, which is used to effect the NCL, can be
turned into Ala amino acid by desulfurization, either after the
ligation step, or after obtaining the thioester peptide, in order
to revert to the native polypeptide structure after ligation.
[0072] Thus, after removal of fragment 2 from the solid support,
the polypeptide C-terminal fragment 2, having a Cys amino acid on
its N-terminal side and a "latent thioester functionality" on its
C-terminal side, would have the structure of general Formula
II:
##STR00004##
wherein R, R.sub.1, R.sub.2 and R.sub.3 are as defined
hereinabove.
[0073] Once this fragment is ligated with fragment 1 (the
N-terminal peptide fragment-thioester) the total polypeptide (for
example, ubiquitin or Ub analog) is obtained, being still linked to
the latent thioester functionality.
[0074] In another embodiment, if the full polypeptide is attached
to the latent thioester functionality, a similar structure would be
obtained, wherein instead of the Cys-peptide fragment, there would
be a Peptide attached to the latent thioester functionality.
[0075] Following the ligation, if necessary, the obtained
polypeptide attached to the latent thioester functionality, can be
reacted with an external thiol under acidic conditions to activate
the removal of the LTF and to obtain the requested polypeptide
thioester.
[0076] It can be seen that the "latent thioester functionality"
attached to the C-terminal side of a peptide or a fragment thereof
(for example as depicted by Formula II) is independent and stable
and can be kept as such until the moment when ligation and/or
activation are required, the LTF group acting as a "switchable
device", there is now provided yet a new aspect of the
invention.
[0077] Thus, according to this additional aspect of the invention,
there is provided a latent thioester functionality (LTF) attached
to the C-terminus of a first polypeptide, or to a first fragment
thereof having a Cys residue at its N-terminus.
[0078] Preferably, the latent thioester functionality has the
general Formula I:
##STR00005##
Wherein:
[0079] R is either hydrogen or a thiol protecting group;
[0080] The thioester functionality being attached to the first Ub
polypeptide fragment via the Nitrogen attached to R.sub.1.
[0081] Further wherein R.sub.1 is selected from the group
consisting of: hydrogen, C1-C3 alkyl, C1-C3 alkyl-COOH, C1-C3
alkyl-CONH.sub.2, C1-C3 alkylene-CONH.sub.2, C1-C3
alkylene-CO.sub.2H, SO.sub.2-alkyl; SO.sub.2-alkyl-CONH.sub.2,
benzyl and derivatives thereof, alkyl-nitrile and
alkyl-halogens;
[0082] Yet further wherein R.sub.2 and R.sub.3 are independently
selected from the group consisting of: hydrogen, CO.sub.2H,
CH.sub.2CO.sub.2H, --CH.sub.2OH, CONH.sub.2CH.sub.2-CONH.sub.2 and
CH.sub.2NH.sub.2, as well as N-protected derivatives thereof.
[0083] Such that at least one of R.sub.1 and R.sub.2 should contain
a linking group CONH.sub.2 or N-protected derivatives thereof.
[0084] Preferably, the latent Thioester Functionality (LTF) is a
residue of N-methyl cysteine.
[0085] Given the importance of modifications in the peptide chain,
the polypeptide contains at least one unnatural amino acid.
Preferably, this unnatural amino acid is a 1,2 thioamine containing
amino acid. More preferably, the 1,2 thioamine containing amino
acid is a protected mercaptolysine. This compound was used for
example in the preparation of tetra-Ub, as demonstrated in parallel
patent application 11-069, as disclosed hereinabove
(Ub3.sub.k48-LTF).
[0086] According to one preferred embodiment, the peptide or
peptide fragment to which the latent thioester functionality is
attached is ubiquitin. However any other LTF-attached polypeptide
combination is included within the scope of this invention.
[0087] The term "thiol" or "thiol compound" as used herein,
represents a group of formula --SH. The term "external thiol"
emphasizez that the thiol group comes from an external reagent,
namely a reagent other than the polypeptide or fragments
thereof.
[0088] Examples of thiol compounds include, but are not limited to,
mercaptoaminomethane, 2-mercapto-1-aminoethane,
3-mercapto-1-aminopropane, 4-mercapto-1-aminobutane,
1,1,1-triamino-2-mercaptoethane, mercaptoacetic acid,
2-mercaptopropionic acid, 3-mercaptobutyric acid (MPA),
4-mercaptovaleric acid and 1,1,1-triamino-3-mercaptopropane.
[0089] Some other exemplary external thiols are shown in scheme 2
below:
##STR00006##
[0090] Preferably, the external thiol used for the final stage in
the afore-mentioned process, is 3-mercaptopropionic acid (MPA).
[0091] The term "acidic conditions" refers to a pH substantially
lower than 7, preferably a pH which is under 6, more preferably
under 4. Most preferably, the necessary acidic pH for "switching
off" the latent thioester functionality, is a pH from about 1 to
about 2.
[0092] It should be noted that for "switching off" the latent
thioester functionality, the acidic conditions can be provided in
one step with the addition of the external thiol, for example when
the thiol is an acid such as MPA. Alternatively, namely, when the
external thiol is not an acid or is not acidic enough, the
conditions should be modified by adjusting the pH to the required
pH described herein.
[0093] As shown in the Experimental section which follows, the
process described hereinabove was successfully applied for the
long-sought chemical preparation of ubiquitin thioesters.
[0094] Thus, according to one aspect of the invention, there is
provided a process for the preparation of ubiquitin thioesters,
this process comprising: [0095] a) Attaching a Latent Thioester
Functionality (LTF) to a solid support; [0096] b) Chemically
synthesizing a ubiquitin monomer or a fragment thereof on this
solid support, followed by removal from the solid support, to
obtain an N-terminal unprotected ubiquitin monomer or an N-terminal
unprotected ubiquitin fragment, attached to the latent Thioester
Functionality on its C-terminal; [0097] c) If a ubiquitin fragment
is obtained in step b: [0098] i. Chemically synthesizing a second
ubiquitin fragment to be complimentary to the ubiquitin fragment
obtained in step b, whereas the second ubiquitin fragment is
prepared as a thioester; [0099] ii. Reacting the thioester of the
second ubiquitin fragment with the ubiquitin fragment attached to
the latent Thioester Functionality obtained in step b, by native
chemical ligation (NCL) to obtain a ubiquitin monomer attached to
the latent Thioester Functionality; [0100] d) Reacting the
ubiquitin monomer being attached to the latent Thioester
Functionality with an external thiol under acidic conditions to
obtain the ubiquitin thioester.
[0101] As used herein, the term "ubiquitin" or Ub includes within
its scope all known as well as unidentified eukaryotic Ub homologs
of vertebrate or invertebrate origin. Examples of Ub polypeptides
as referred to herein include the human Ub polypeptide that is
encoded by the human Ub encoding nucleic acid sequence (GenBank
Accession Numbers: U49869, X04803) as well as all equivalents.
[0102] For example, natural human Ub protein has the following
sequence, containing the following 76 amino acids:
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSD
YNIQKESTLHLVLRLRGG. (SEQ ID NO: 1).
[0103] Therefore, according to one preferred embodiment, the
ubiquitin polypeptide is a natural ubiquitin polypeptide.
[0104] However, as used herein, the term "ubiquitin" (Ub) also
includes modified ubiquitin polypeptides.
[0105] The term "modified Ub" as used herein refers to polypeptides
containing one or more unnatural amino acids replacing one or more
of the 76 native Ub amino acids.
[0106] For example, in the ensuing examples, an equivalent sequence
to natural Ub was synthetically prepared, replacing the Met amino
acid with a Leucine amino acid (namely to obtain the following
sequence:
LQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSD
YNIQKESTLHLVLRLRGG) (SEQ ID NO: 2), thereby avoiding oxidation of
the Met. Similarly, another equivalent is obtained by replacing the
Met with nurleucine (Nle). However, the invention also works with
the original Met amino acid.
[0107] Additional examples to modified Ub include replacing Leu
amino acids in positions 28 or 46 by a 1,2 thioamine containing
amino acid, such as mercaptolysine derivatives (as shown in Example
7 below), or by introducing a labeled amino acid, or an amino acid
linked to a specific reagent etc. Additional useful modifications
can be envisioned by a person skilled in the art and are therefore
included in the scope of this invention.
[0108] Thus, according to one preferred embodiment, the ubiquitin
polypeptide is a modified ubiquitin polypeptide.
[0109] The ubiquitin according to the present invention includes
both mono-ubiquitin and poly-ubiquitin. In other words, the
ubiquitin may appear as having either one or several ubiquitin
monomers.
[0110] The term "Ub monomer", used interchangeably with the term
"Ub unit", as used herein, refers to a 76-amino acid sequence of
ubiquitin, either natural or modified.
[0111] Furthermore, this term includes ubiquitin-like-modifiers
(ULM), also termed "ubiquitin-like" or "Ubl" protein modifiers.
This terms, as used herein, refers to the group of small proteins
that are subject to conjugation machinery similar to that for
ubiquitination. Examples of Ubl protein modifiers include NEDD8,
ISG15, SUMO1, SUMO2, SUM03, APG12, APG8, URM1, Atg8, URM1, HUB1,
FUB1, FAT10, UBL5, UFM1, MLP3A-LC3, ATG12, as well as other Ubl
protein modifiers yet to be identified.
[0112] The process for preparing Ub thioesters, is disclosed in
detail hereinabove as part of the general discussion on preparing
polypeptide thioesters according to the present invention, whereas
the terms "polypeptide" or "peptide" therein should be read to
refer to ubiquitin polypeptides. All other terms are as described
hereinabove.
[0113] While the process described herein can be conducted by
"elongating" one long chain of the Ub monomer/unit on the solid
support, to which the LTF is attached, this process is less
desirable for the 76-amino acid-long ubiquitin, having lower yields
and is generally less convenient, since any modification in this
long chain requires a complete synthesis of the entire 76-amino
acid chain . . . . Therefore, although complete synthesis of Ub
thioesters has been demonstrated in the Examples below, using
native chemical ligation (NCL) of shorter fragments of the Ub
monomer is a preferred embodiment of the present invention.
[0114] Since one preferable way of conducting NCL is based on a
reaction of a thioester fragment with a Cys amino acid on the
second peptide fragment, and since the natural Ub sequence has no
Cys amino acids, NCL of ubiquitin fragments is preferably conducted
in the positions containing Ala amino acids (namely positions 28
and 46), by chemically introducing one or more Cys amino acids into
one or more of those positions, whereas at some stage after the
ligation, the Cys is turned back into native Ala by
desulfurization.
[0115] For example, the process described herein can be performed
wherein the ubiquitin monomer is prepared of two ubiquitin segments
by NCL, such that the fragment attached to the LTF is:
AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub46-76) (SEQ ID NO: 3) and the
second fragment being in its thioester form is
LQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIF (Ub1-45), (SEQ ID NO:
4) whereas the A.sub.46 amino acid is temporarily replaced by
Cysteine.
[0116] Another option for preparing the Ub of two ubiquitin
segments is wherein the fragment attached to the LTF is:
AKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGG (Ub28-76) (SEQ ID
NO: 5) and the second fragment, being in its thioester form, is
LQIFVKTLTGKTITLEVEPSDTIENVK (Ub1-27), (SEQ ID NO: 6) whereas the
A.sub.28 amino acid is temporarily replaced by Cysteine.
[0117] The ubiquitin can further be ligated from three fragments.
In this case, the process described herein needs to be somewhat
modified as follows:
[0118] First, both (Ub46-76) and (Ub28-45) (AKIQDKEGIPPDQQRLIF (SEQ
ID NO: 7) and AGKQLEDGRTLSDYNIQKESTLHLVLRLRGG, (SEQ ID NO: 3)
respectively) fragments are separately prepared on solid supports,
wherein both the A.sub.46 and the A.sub.28 amino acids are
temporarily replaced by Cysteine, each being attached to an LTF
group, as described above according to the embodiments of the
present invention, and are removed from these supports.
[0119] Then, Ub(1-27) (LQIFVKTLTGKTITLEVEPSDTIENVK) (SEQ ID NO: 6)
fragment is separately prepared on a solid surface, removed and
turned into a thioester to obtain Ub(1-27)-SR.
[0120] NCL OF the two fragments Ub(1-27)-SR and the modified
C28-Ub(29-45)-LTF is conducted to obtain the Ub(1-45), still
attached to the LTF group. Following activation under acidic
conditions and a reaction with an external thiol, the Ub1-45
thioester is obtained.
[0121] Finally, NCL OF C46-Ub(47-76)-LTF and (UB 1-45)-SR follows,
as conducted in the ligation of the two Ub fragments.
[0122] As shown in Example 5, the inventors have shown that the
synthetic ubiquitin thioester obtained by this method has a similar
behavior in peptide ubiquitylation as the ubiquitin thioester
obtained via gene expression.
[0123] The newly developed process is advantageous in allowing
higher flexibility in the chemical manipulation of ubiquitin
thioesters in a wide variety of ubiquitylated peptides and proteins
for structural and biochemical analysis and for the synthesis of
ubiquitin chains.
[0124] Indeed, the successful application of this method in the
preparation of poly-Ub chains is described in a patent application
being co-filed on the same date as the instant application, having
the reference number 11-069, claiming the same priority
(provisional application No. 61/302,359) and entitled "Chemical
Preparation Of Polyubiquitin Chains", which is incorporated by
reference as if fully set forth herein.
[0125] It should be noted that although the examples and
description provided herein are based on the ligation and
corresponding fragments as described hereinabove, the ligation can
be conducted between other ligation sites, and the fragments would
be chosen according to the requirements of this other ligation
process, as known to a person skilled in the art, without changing
the scope of the invention.
[0126] One specific embodiment of the invention for obtaining Ub-SR
is provided in Scheme 3 below and in Examples 3 and 4. This process
is based on the synthesis of the Ub monomer from two fragments,
which include peptide 1 Ub(46-76) and peptide 2 Ub(1-45)-SR,
wherein Ala.sub.46 is mutated temporarily to Cys to facilitate NCL,
bearing in mind that this Cys could be converted to Ala using the
desulfurization reaction. To achieve the desired C-terminal
functionality, peptide 1 is equipped with N-methylcysteine, as the
N-S acyl transfer device (latent thioester functionality), in which
the thiol side chain is protected with a photolabile-protecting
group (2-nitrobenzyl) to avoid intramolecular N-S acyl transfer in
the TFA-cleavage step. Upon completion of the ligation reaction,
the thiol-protecting group is removed by UV, followed by activation
of the fully unprotected polypeptide under acidic conditions, and
treatment with MPA to afford the Ub-SR.
##STR00007##
[0127] Thus, according to another aspect of the invention, there is
provided a process for the preparation of ubiquitin thioesters,
this process comprising: [0128] a; Chemically synthesizing a first
ubiquitin polypeptide fragment, being attached to a Latent
Thioester Functionality (LTF) on the C-terminal of the ubiquitin
fragment, further wherein this ubiquitin fragment contains an
unprotected Cysteine amino acid on the N-terminal side thereof;
[0129] b) Chemically synthesizing a second ubiquitin fragment being
complimentary to the first ubiquitin fragment obtained in step a,
wherein this second ubiquitin is in a thioester form; [0130] c)
Ligating the first ubiquitin fragment with the second ubiquitin
fragment, to obtain an unprotected ubiquitin polypeptide attached
to the latent Thioester Functionality; [0131] d) Reacting the
ubiquitin polypeptide attached to the latent Thioester
Functionality with an external thiol under acidic conditions to
obtain the Ubiquitin thioester;
[0132] Optionally, the process described herein further comprises
desulfurization of the Ubiquitin thioester to turn the unnatural
Cys amino acid into an Ala amino acid, either before or after step
(d).
[0133] Following this successful process, kits and apparatus for
assembling polypeptide thioesters by the processes described herein
are also provided.
[0134] Therefore, according to yet another aspect of the invention,
there is provided a kit for preparing assembled polypeptide
thioesters comprising: [0135] (a) a latent thioester functionality,
as described hereinabove, attached to the C-terminus of a first
polypeptide or a fragment thereof, this fragment having a Cys
residue at its N-terminus; [0136] (b) optionally (if container (a)
has only a fragment of the desired polypeptide) , a second
container containing a second polypeptide fragment having at its
N-terminus a thioester; wherein the N-terminal cysteine of the
first polypeptide fragment attached to the latent thioester
functionality, is capable of selectively ligating to the N-terminus
of the second polypeptide fragment, to form a polypeptide
comprising the latent thioester functionality at its C-terminus;
and [0137] (c) one or more additional containers containing an
activating acid and an external thiol, capable of reacting with the
polypeptide comprising a latent thioester functionality at its
C-terminus, to provide a polypeptide thioester, wherein the acid
and the thiol may be the same compound, or if different, may be
provided either separately or in the same container.
[0138] Additional additives may be added to each of these
container, or provided separately, in order to facilitate the
ligation, the activation of the LTF group, or the obtaining of the
final Ub thioester.
[0139] Preferably, the polypeptide thioester prepared using this
kit is a ubiquitin thioester.
[0140] Preferably, the solid support is a bead resin.
[0141] Preferably, the Ub polypeptides or fragments thereof are all
prepared by Solid phase peptide synthesis (SPPS).
[0142] According to a preferred embodiment, at least one of either
polypeptide fragment (a), polypeptide fragment (b) or the complete
polypeptide comprising the kit, contains an unnatural amino
acid.
[0143] Preferably the unnatural amino acid is a 1,2 thioamine
containing amino acid.
[0144] The term "1,2 thioamine containing amino acid" refers to
amino acids containing the 1,2 thioamine group. Examples of 1,2
thioamine containing amino acids include, but are not limited to,
mercaptolysine and various modifications thereof, as well as to the
products obtained from the reaction of Cys amino acid with one of
the following: glutamic acid, aspartic acid, Ser, Thr and Lys.
[0145] Preferably, the 1,2 thioamine containing amino acid is a
protected mercaptolysine. Most preferably, the protected
mercaptolysine is thiazolidine (Thz)-protected mercaptolysine.
[0146] The preparation of peptide fragment 1 was accomplished
according to the sequence of reactions shown in Scheme 4 and
detailed in Example 1.
[0147] Initially, the latent thioester functionality is attached to
the solid support.
[0148] It is important to note that the attachment of the Latent
Thioester Functionality (LTF) to the solid support may be conducted
in one or several steps. For example, as shown in Scheme 4 and
detailed in Example 1, a Rink amide resin was loaded with Fmoc-cys
(2-nitrobenzyl) --OH using HBTU/DIEA coupling conditions.
Subsequently, the Fmoc-protecting group was removed with 20%
piperidine followed by coupling of the free amine with
o-nitrobenzenesulfonyl chloride (o-NBS) to facilitate
N-methylation. Selective deprotonation of the sulfonamide with DBU
and alkylation with methyl p-nitrobenzenesulfonate in DMF led to
the formation of the methylated sulfonamide resin 6. Alternatively,
it was found that TBAF/Mel could also serve as an excellent choice
for the methylation step. Selective removal of the o-NBS was
achieved by using mercaptoethanol and DBU in DMF to obtain the
N-methyl-cysteine, acting as LTF according to the present
invention, being attached to the solid support.
[0149] Subsequently, SPPS was conducted, from amino acid (G) to
amino acid (C), to obtain the desired peptide, the C-terminal of
which being attached to the N-methyl-cysteine LTF, which is on its
other side attached to the solid support throughout the SPPS. Side
chain deprotection and release from the solid support using
TFA/TIS/H.sub.2O (95:2.5:2.5) afforded, after RP-HPLC purification,
the desired peptide (Cys-Ub (47-76) --N-methyl Cysteine) in 25-30%
isolated yield, whereas the obtained peptide has an unprotected Cys
amino acid on its N-terminal, still attached to the LTF group on
its C-terminal side, quite unlike common linkers in SPPS, which are
usually detached either when the peptide is released from the
resin, or during ligation under ligation conditions.
##STR00008##
[0150] In one additional embodiments, this synthesis was continued
in full to obtain the entire Ub polypeptide by SPPS, with the
modification that the thiol protecting group on the LTF was trityl.
In another embodiment, the full synthesis was repeated, while
replacing the K amino acid at position 48 with a Thz-protected
mercaptolysine. In both cases, final release from the solid support
using TFA/TIS/H.sub.2O (95:2.5:2.5), followed by reaction with MPA
at a pH ranging from 1 to 2.
[0151] For the synthesis of Ub polypeptide fragment 2 (as it
appears in Scheme 3) Ub(1-45)--SR, (R=--CH.sub.2CH.sub.2-COOMe), it
was chosen to apply the N-acylurea chemistry, as is shown in Scheme
5 below and as detailed in Example 2.
##STR00009##
[0152] As can be seen in Scheme 5 and in Example 2, SPPS was
conducted using HBTU/DIEA coupling conditions on a rink resin,
whereas the first amino acid being 3-Fmoc-4-damino benzoic acid
(Fmoc-Dbz), a residue necessary for subsequent thioester formation,
followed by Phe as the first Ub peptide fragment residue, and the
last amino acid being coupled in the Boc protected form. On resin
activation was conducted by adding p-nitrophenylchloroformate. The
resin was washed with CH.sub.2Cl.sub.2 adding DIEA in DMF to obtain
the peptide with the N-acyl benzimidazolinone functionality. This
peptide was deprotected and cleaved from the resin by treatment
with a mixture of TFA/H.sub.2O/TIS (95:2.5:2.5). After the
lyophilization step, the crude peptide was treated with methyl
3-mercaptopropionate in 6 M Gn.HCl, pH 7 to afford the Ub(1-45)-SR
2, after RP-HPLC purification step, in 20% yield.
[0153] As shown in Scheme 3, and as detailed in Example 3, the
ligation between Ub peptide fragments 1 and 2 was carried out under
NCL conditions i.e. 6 Gn.HCl, 200 mM phosphate buffer, pH 7.5 in
the presence of 2% (v/v) thiophenol/benzyl mercaptan. The reaction
was followed by HPLC and mass spectrometry, which indicated nearly
a complete ligation after 8 hours. As detailed in Example 4,
following purification and lyophilization steps, when the thiol was
protected by a photo-labile protecting group, the product was first
exposed to UV light (365 nm) for 2 hours. The final step was the
addition of 20% (v/v) MPA at at pH 2 and the reaction mixture was
left at 37.degree. C. After 12 hours a full conversion to the
desired thioester product was achieved. Preparative RP-HPLC
purification and lyophilization afforded the Ub-SR in 30% isolated
yield (for two steps) .
[0154] To give further support of the integrity of the C-terminal
thioester functionality, the synthetic Ub-SR was tested, as
detailed in Example 5, in peptide ubiquitylation using
.alpha.synuclein (1-17) model peptide bearing the mercaptolysine
residue. The results show that the synthetic Ub-SR is indeed an
excellent substrate in the ligation reaction wherein within 4 hours
a complete reaction was observed to afford the ubiquitylated
peptide in 60% isolated yield. Furthermore, the ligation product
was desulfurized using metal free desulfurization conditions to
convert the Cys to Ala along with the full removal of the thiol
handle from the mercaptolysine to furnish the ubiquitylated peptide
8 (Scheme 6). The desulfurized product was isolated in 75% yield
and was treated with ubiquitin C-terminal hydrolase, UCH-L3 for 12
hours. The results show that the desulfurized product is indeed
UCH-L3 substrata affording both the hydrolyzed Ub and the
.alpha.-syn (1-17). Thus, the inventors have been able to show that
the synthetic Ub-SR is in analogy to the E1-E2 activation steps and
when combined with the ubiquitylation step using mercaptolysine,
which resemble the E3 ligase activity, shows that the entire
ubiquitylation process could be mimicked using chemical tools
only.
##STR00010##
[0155] Thus, the inventors have successfully proved that
Ub-thioesters can be chemically synthesized, and have the same
biological activity as the natural Ub-thioesters.
[0156] Furthermore, the authors have previously reported a new
method for highly efficient and chemoselective peptide
ubiquitylation utilizing .delta.-mercaptolysine residue, and have
suggested that isopeptide formation assisted by
.delta.-mercaptolysine is reminiscent to amide bond formation via
NCL, which includes a capture step of the ubiquitin thioester
(Ub-SR) to form a transient thioester intermediate that
spontaneously rearranges through S-N acyl transfer step to form the
isopeptide bond. To enable the incorporation of the
.delta.-mercaptolysine residue, without it being unmasked during
ligation or during Boc- or Fmoc-SPPS, the authors realized that
protecting group variations of this residue are required. As can be
seen below, the authors have now successfully devised a general
strategy for the synthesis of different analogues of the
.delta.-mercaptolysine (for example, compounds 1b-e, Scheme 7, vs.
unprotected .delta.-mercaptolysine, 1a in this Scheme) bearing a
variety of protecting groups on the .alpha.- and .epsilon.-amine,
as well as on the .delta.-thiol paving the way for the use of these
analogues in the synthesis of ubiquitylated proteins.
##STR00011##
[0157] While mercaptolysine derivative 1a could be incorporated in
peptides and peptide thioesters using Boc-SPPS, after the removal
of alloc under palladium catalysed reaction condition, treatment
with HF or TFMSA would release all protecting groups from the
mercaptoamine moiety. On the other hand, 1e under similar cleavage
conditions (i.e., HF/TFMSA) would retain the mercapto functionality
protected, thus making it useful in sequential ligation.
Mercaptolysine derivatives 1b-d could be installed in peptides
using Fmoc-SPPS. Again, while 1c, the equivalent to 1a, is
completely unmasked under the cleavage conditions, 1b and 1d could
be used in sequential ligation due to the orthogonality of the
protecting groups (i.e., thiazolidine (Thz) and
P-hydroxymercuribenzoate (PMB)).
[0158] Of particular interest is the thiazolidine (Thz)-protected
mercaptolysine amino acid 1b, which can be easily turned into
nucleophilic amine upon reaction with methoxylamine under acidic pH
(of about 4).
[0159] This amino acid can be incorporated as a building-block in
the structure of ubiquitin or other polypeptides, by replacing the
similarily-structured Lys amino acid during SPPS of one or more
fragments, as described herein. When this replacement is done
during the preparation of a ubiquitin thioester, according to the
present invention, there is obtained a ubiquitin thioester
containing a protected mercaptolysine "handle", which can then be
used in a variety of applications, such as sequential ligation, for
example for preparing poly-ubiquitin chains, as disclosed in a
patent application being co-filed on the same date as the instant
application, having the reference number 11-069PCT, claiming the
same priority (provisional application No. 61/302,359) and entitled
"Chemical Preparation Of Polyubiquitin Chains", which is
incorporated by reference as if fully set forth herein.
[0160] Thus, according to another aspect of the invention, there is
provided a tiazolidine (Thz)-protected mercaptolysine amino
acid.
[0161] According to a preferred embodiment, this amino acid serves
as a building block for the ubiquitin thioester described
hereinabove.
[0162] The term "building-block" as used herein refers to an amino
acid which is incorporated in the sequence of the desired
polypeptide, or polypeptide thioester, for example an amino acid
which is incorporated in the sequence of the desired ubiquitin
thioester, according to the present invention. The building block
can be incorporated both on the main backbone of the polypeptide,
as well as on a side chain thereof.
[0163] Preferably, the tiazolidine (Thz)-protected mercaptolysine
amino acid described herein has the general formula III:
##STR00012##
[0164] Wherein Ra and Rb are independently either hydrogen or
nitrogen protecting groups.
[0165] Examples of amine protecting groups include, but are not
limited to, the following: 1) acyl types such as formyl,
trifluoroacetyl , and p-toluenesulfonyl; 2) aromatic carbamate
types such as benzyloxycarbonyl (Cbz) and substituted
benzyloxycarbonyls, 1-(p-biphenyl)-1-methylethoxycarbonyl, and
9-fluorenylmethyloxycarbonyl (Fmoc); 3) aliphatic carbamate types
such as tert-butyloxycarbonyl (Boa) , di-tert-butyl dicarbonate
(Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and
allyloxycarbonyl; and 4) cyclic alkyl carbamate types such as
cyclopentyloxycarbonyl and adamantyloxycarbonyl. Other Amine
protecting groups are known to a person skilled in the art.
[0166] Preferably, Ra and Rb are either hydrogen or the protecting
groups Boc or Fmoc.
[0167] The 1,2 thioamine containing amino acids disclosed
hereinabove were prepared by first using nitro olefin 2 as the
crucial building block, which allows the incorporation of the thiol
functionality through 1,2-addition reaction at a high efficiency,
as seen in Scheme 8 below. The obtained precursor 3b was used for
the synthesis of other analogues bearing different protecting
groups on the thiol functionality through the use of various thiol
nucleophiles Scheme XX).
##STR00013##
[0168] Thus, the 1,2-addition of trityl thiol, PMB-SH, acetamido
methyl thiol and t-BuSH on 2 under NaHMDS/n-BuLi conditions
furnished 3a-d in 75-85% yield. It should be noted that these
reactions occurred in non-stereoselective fashion resulting in both
isomers in a nearly equal ratio. However, the desulfurization
reaction, which follows the ligation step, removes this
diastereomeric center affording only single peptidic isomer.
[0169] In designing the synthesis of 1b-c, it was established that
the synthesis of these analogues could be accomplished from the
common intermediate 4 which can be obtained from 3b by replacing
the di-boc to Fmoc protection (Scheme 3). Precursor 4 was converted
to 5 through sequence of reactions, which included reduction of the
nitro group using Zn/HCl, deprotection of the trityl group,
incorporation of thiazolidine, and N-Boc protection. Subsequent
hydrolysis of the methyl ester 5 yielded the target molecule 1b in
84% yield. For the synthesis of 1c, intermediate 4 was initially
subjected to a saponification step to give the acid derivative 6,
that on reduction of the nitro group and N-Boc protection afforded
the desired amino acid 1c (Scheme 9).
##STR00014##
Whereas:
[0170] step a includes [0171] i) HCl(g), ethylacetate (dry) ,
-20.degree. C., 1 hour, and [0172] ii) Fmoc-OSu, NaHCO.sub.3,
Dioxane-water, RT, 2 hours; [0173] Step b includes: [0174] i) Zn
(dust), 10% HCl, 2 hours, RT; [0175] ii) TFA, TIS,
CH.sub.2Cl.sub.2, RT, 30 minutes, [0176] iii) HCHO, NaHCO.sub.3,
MeOH, rt, 15 hours., and [0177] iv) (Boc).sub.2O, RT, 15 hours;
[0178] Step c includes: [0179] i) LiOH, THF-H.sub.2O (4:2),
0.degree. C., 1 hour, [0180] Step d includes [0181] i) LiOH,
THF-H.sub.2O (4:1), 0.degree. C., 50 minutes; and [0182] Step e
includes: [0183] i) Zn (dust), 10% HCl, 2 hours, RT; and [0184] ii)
(Boc)).sub.2O, MeOH-TEA (10:1), 2 hours.
[0185] The conjugate addition product 3c was used for the synthesis
of mercaptolysine analogue 1d in 5 steps process wherein the di-boc
protecting group was first replaced with the Fmoc to yield
precursor 7. The latter was reduced using NiCl.sub.2/NaBH.sub.4
conditions followed by protection using Boc-anhydride to give 8,
which was subjected to a saponification step to afford the desired
analogue 1d (Scheme 4). For the synthesis of 1e, the nitro group in
3d was reduced to the amine under NiCl.sub.2/NaBH.sub.4 conditions
followed by protection with Z-(2-Cl)-OSu to give 9. Subsequently,
the di-boc was switched to the mono-boc to afford 10, which was
hydrolyzed to the target molecule 1e (Scheme 10).
##STR00015## [0186] Step a includes: [0187] i) HCl(g), ethylacetate
(dry) , -20.degree. C., 1 hour, and [0188] ii) Fmoc-OSu,
NaHCO.sub.3, Dioxane-water, RT, 2 hours; [0189] Step b includes:
[0190] i) NaBH.sub.4, NiCl.sub.2.6H.sub.2O, THF-MeOH (1:1), -20 to
-15.degree. C., 35 minutes, and [0191] ii) (Boc).sub.2O, MeOH-TEA
(10:1), 2 hours; [0192] Step c includes: [0193] i)LiOH,
THF-H.sub.2O (4:2), 0.degree. C., 1 hour; [0194] Step d includes:
[0195] i) NaBH.sub.4, NiCl.sub.2.6H.sub.2O, THF-MeOH (1:1), -20 to
-15.degree. C., 20 minutes, [0196] ii) Z-(2-Cl)-OSu, NaHCO.sub.3,
Dioxane-water, RT, 2 hours; [0197] Step e includes: [0198] i)
HCl(g), ethylacetate (dry), -20.degree. C., 1 hour, [0199] ii)
(Boc).sub.2O, MeOH-TEA (10:1), 2 hours; [0200] Step f includes:
[0201] i) LiOH, THF-H.sub.2O (4:2), 0.degree. C., 1 hour.
[0202] As explained hereinabove, the 1,2 thioamine substituents on
Ub thioesters are important as sources for nucleophilic handles in
reactions using the Ub thioesters. Therefore, according to a
preferred embodiment of the invention, the ubiquitin thioesters
described herein further contain at least one 1,2 thioamine
containing amino acid.
[0203] Preferably, this 1,2 thioamine containing amino acid is a
protected mercaptolysine. Most preferably, this protected
mercaptolysine is thiazolidine (Thz)-protected mercaptolysine.
[0204] As seen in Example 7, the processes described in Examples
1-4 were repeated, with the only modification being the usage of
unnatural amino acids (in this example 1,2 thioamine containing
amino acid) in various stages of the process, instead of one of the
seven natural lysines in ubiquitin (their natural positions being
K6, K11, K27, K29, K33, K48, K63).
[0205] In particular, if the amino acid to be replaced is the Lys
in positions 48 or 63, the modification is done during the
preparation of fragment LTF-UbC, according to Example 1, by
replacing the requested lysine by the 1,2 thioamine containing
amino acid.
[0206] On the other hand, if the amino acid to be replaced is the
Lys in positions 6, 11, 27, 29 or 33, the modification is done
during the preparation of fragment UbN-SR, according to Example 2,
by replacing the requested lysine by the 1,2 thioamine containing
amino acid.
[0207] Therefore, according to yet another aspect of the present
invention, there is provided a ubiquitin thioester comprising at
least one ubiquitin monomer, this ubiquitin thioester containing at
least one 1,2 thioamine containing amino acid. Preferably, the 1,2
thioamine containing amino acid is a protected mercaptolysine .
Most preferably, the protected mercaptolysine is thiazolidine
(Thz)-protected mercaptolysine.
[0208] According to additional preferred embodiments of the present
invention, the Ubiquitin thioester described herein has the general
formula II:
##STR00016##
wherein Ubm is a ubiquitin chain having m ubiquitin monomers, m
being an integer equal to or larger than 1, R being selected from
either alkyls or aryls, said alkyls or aryls being optionally
substituted, and Ap being said 1,2 thioamine containing amino
acid.
[0209] The term Ub chain as used herein refers to both mono-Ub and
poly-Ub, namely ubiquitins having 1 or more monomers, as those have
been defined hereinabove.
[0210] Preferably, the 1,2 thioamine containing amino acid is a
protected mercaptolysine. Most preferably, the protected
mercaptolysine is thiazolidine (Thz)-protected mercaptolysine.
[0211] Additional objects, advantages, and novel features of the
present invention will become apparent to one ordinarily skilled in
the art upon examination of the following examples, which are not
intended to be limiting. Additionally, each of the various
embodiments and aspects of the present invention as delineated
hereinabove and as claimed in the claims section below finds
experimental support in the following examples.
EXAMPLES
[0212] Reference is now made to the following examples, which
together with the above descriptions, illustrate the invention in a
non limiting fashion.
MATERIALS AND ANALYTICAL METHODS
Materials
[0213] The meaning of the abbreviations used in the description and
the claims is as outlined in the table below: [0214] Fmoc
9-Fluorenylmethoxycarbonyl-- [0215] Boc t-Butoxycarbonyl-- [0216]
DIEA Diisopropylethylamine [0217] TFA Trifluoraceticacid [0218] DMF
N,N'-Dimethylformamide [0219] HBTU O-Benzotriazole
N,N,N',N'-tetramthyl-uronium-- [0220] HOBt 1-Hydroxybenzotriazole
[0221] DBU 1, 8-Diazabicyclo [5.4.0]undec-7-ene [0222] HATU
O--(7-Azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-hexafluoro-phosp-
hate [0223] MEI Methyl iodide [0224] TBAF Tetrabutylammonium
Fluoride [0225] MPA 3-mercaptopropionic acid [0226] DTT
Dithiothreitol [0227] Tris Tris-(hydroxymethyl) aminomethane [0228]
THF Tetrahydrofuran [0229] NaHMDS Sodium Hexamethyldisilazane
[0230] TIS Triisopropylsilane
[0231] DMF was purchased in biotech grade. Commercial reagents were
used without further purification.
[0232] Resins, protected and unprotected amino acids, and coupling
reagents (HBTU, HOBt) were purchased from Novabiochem.
[0233] Buffer B is acetonitrile with 0.1% v/v TFA and buffer A is
water with 0.1% v/v TFA.
[0234] Methanol, triethylamine, THF, ether were purified and dried
before use.
[0235] The n-hexane used was the fraction distilling between
40-60.degree. C.
[0236] Natural ubiquitin, which was used for comparison (from
bovine erythrocytes) was purchased from Sigma.
[0237] All other chemicals were purchased from either Aldrich
and/or Fluka.
[0238] SPPS was carried out manually in syringes, equipped with
teflon filters, purchased from Torviq or by using an automated
peptide synthesizer (CS336X, CSBIO). If not differently described,
all reactions were carried out at room temperature.
Note: Throughout this specification amino acid residues will be
denoted by the three-letter abbreviation or single-letter code as
follows:
Three-Letter One-Letter Amino Acid Abbreviation Symbol
[0239] Alanine Ala A [0240] Arginine Arg R [0241] Asparagine Asn N
[0242] Aspartic Acid Asp D [0243] Cysteine Cys C [0244] Glutamine
Gin Q [0245] Glutamic Acid Glu E [0246] Glycine Gly G [0247]
Histidine His H [0248] Isoleucine lie I [0249] Leucine Leu L [0250]
Lysine Lys K [0251] Methionine Met M [0252] Phenylalanine Phe F
[0253] Proline Pro P [0254] Serine Ser S [0255] Threonine Thr T
[0256] Tryptophan Trp W [0257] Tyrosine Tyr Y [0258] Valine Val
V
Instrumental Data:
[0259] Mass spectrometry was conducted using LCQ Fleet Ion Trap
(Thermo Scientific).
[0260] Analytical RP-HPLC was performed on a Thermo instrument
(Spectra System p4000) using an analytical column (Jupiter 5
micron, C18, 300A 150.times.4.6 mm) and a flow rate of 1.2
ml/minute.
[0261] Preparative RP-HPLC was performed on an ECOM instrument
using a preparative column (Jupiter 5 micron, C18, 300A,
250.times.10 mm) and a flow rate of 25 mL/minute.
EXAMPLE 1
Preparation of N-methylcysteine Peptide Fragment 1 Ub(46-76)
(LTF-UbC Fragment):
[0262] This example corresponds to Scheme 4.
[0263] In order to prepare a ubiquitin fragment containing a
latent-Thioester-Functionality (LTF), a photolabile protected
N-methyl-cysteine was first attached to the solid substrate as
follows: Cys (2-nitrobenzyl) --OH was coupled to Rink amide resin
(0.56 mmol/g; 0.1 mmol scale) using HBTU in 5 fold excess of the
initial loading of the resin and DIEA was used in 10 fold excess.
Peptide coupling was performed for 30 minutes. Fmoc deprotection
was achieved by treatment of the resin with 20% piperidine.
[0264] Sulfonylation: Collidine (264 .mu.L, 20 eq) was dissolved in
1.5 mL of CH.sub.2Cl.sub.2 and added to the resin, followed by the
addition of solution of o-nitrobenzenesulfonyl chloride (442
mgrams, 20 eq) in 1.5 mL of CH.sub.2Cl.sub.2. The resin was shaken
for 2 hours at RT and was washed using CH.sub.2Cl.sub.2 (3.times.5
mL), and DMF (3.times.5 mL).
[0265] Alkylation: To the washed resin from previous step, DBU (74
.mu.L, 5 eq) in 1.5 mL of DMF was added followed by the addition of
methyl 4-nitrobenzenesulfonate (108 mgrams, Seq) in 1.5 mL of DMF.
The resin was shaken for 1 hour at RT and was washed using DMF
(3.times.5 mL). Alternatively, Mel (124 .mu.L, 20 eq) in 1 ml TBAF
was added to the resin and was shaken for 1 hour.
[0266] Removal of NBS: To a suspension of previously treated resin,
DBU (38 .mu.L, 5 eq) and mercaptoethanol (35 .mu.L, 10 eq) were
added in DMF and shaken well for 30 minutes at RT followed by DMF
wash (3.times.5 mL).
Solid Phase Protein Synthesis (SPPS):
[0267] The first amino acid (Gly) was coupled using HATU (4 eq) and
DIEA (10 eq) for 45 minutes (2x). The remaining amino acids were
coupled using peptide synthesizer.
[0268] Cleavage from the resin: A mixture of TFA,
triisopropylsilane and water (95:2.5:2.5) was added to the dried
peptide-resin and the reaction mixture was shaken for 2 hours at
RT. The resin was removed by filtration and was washed with TFA
(2.times.2 mL). To precipitate the peptide the combined filtrate
was added drop-wise to 10 fold volume of cold ether,
centrifugation, decanting of ether, followed by dissolution of
residue in acetonitrile-water and HPLC purification afforded the
corresponding peptide fragment 1 (according to Scheme 3) in 25-30 %
yield.
[0269] In another experiment, the synthesis was continued in full
to obtain the entire Ub polypeptide by SPPS, with the modification
that the thiol protecting group on the LTF was trityl. Final
release from the solid support using TFA/TIS/H.sub.2O (95:2.5:2.5)
afforded the corresponding full-Ub peptide in 80% crude yield and
20-30 % pure yield.
EXAMPLE 2
Preparation of Ub Fragment 2 as Thioester (UbN-SR Fragment):
[0270] This example corresponds to Scheme 5.
[0271] Rink amide resin (0.2 mmol/grams, 0.1 mmol scale) was used
for the synthesis of UbN-SR. Amino acids and HOBT/HBTU were used in
4-fold excess of the initial loading of the resin. DIEA was used in
10 fold excess. Fmoc deprotection was achieved by treatment of the
resin with 20% piperidine. The first two amino acids, (i.e.
3-Fmoc-4-damino benzoic acid (Fmoc-Dbz), and Phe), were each double
coupled manually for 1 hour. The remaining amino acids were coupled
using peptide synthesizer. The last amino acid was coupled in the
Boc protected form.
[0272] On resin activation: After peptide elongation the resin was
washed with CH.sub.2Cl.sub.2 and a solution of
p-nitrophenylchloroformate (100 mgrams, 5 eq) in 10 ml of
CH.sub.2Cl.sub.2 was added and shaken for 1 hour at RT. The resin
was washed with CH.sub.2Cl.sub.2 (3.times.5 mL) , and DMF
(3.times.5 mL). To the washed resin a solution of 0.5 M DIEA in DMF
(5 mL) was added and shaken for additional 30 minutes. The resin
was washed using DMF (3.times.5 mL).
[0273] Cleavage and purification: The procedure used for peptide 1
was followed.
[0274] Thioesterification: The pure peptide was dissolved in 0.2 M
phosphate buffer (pH 7.98) containing 6 M guanidine.HCl to a final
concentration of .about.1 mM, followed by the addition of 2% (v/v)
methyl-mercaptopropionate. The solution was kept at RT for 1 hour
and purified by preparative reverse-phase HPLC using a linear
gradient of 10-60% B over 30 minutes (buffer A: 0.1% TFA in water;
buffer B: 0.1% TFA in acetonitrile) to afford the corresponding
thioester 2 (numbering according to Scheme 3) in .about.80% crude
yield and 35% pure yield.
EXAMPLE 3
Native Chemical Ligation of Cys-UbC-LTF (1) and UbN-SR (2):
[0275] This example corresponds to Scheme 3.
[0276] The ligation of unprotected peptide segments was performed
following a known procedure: 3.2 mg of UbC and 5 mg of UbN-SR (1.1
eq) were dissolved in 440 .mu.L, of 0.2 M phosphate buffer (pH
7.98) containing 6 M guanidine.HCl to a final concentration of 2
mM. 2% (v/v) thiophenol and benzylmercaptan (8.7 L) were added, and
the ligation reaction was performed in a heating block at
37.degree. C. The reaction was monitored using reverse-phase HPLC
analysis on a C4 column using a linear gradient (10-60% B) over 30
minutes and purified on preparative HPLC using the similar gradient
to obtain the ligation product 3 in a 36% yield.
EXAMPLE 4
[0277] Removal of thiol-protecting group from ligation product, and
treatment with an external thiol:
[0278] This example corresponds to Scheme 3.
[0279] Peptide 3, obtained in Example 3, was dissolved in
photolysis buffer containing 10 mM ascorbic acid; 10 mM
semicarbazide and 10 mM MPA in 0.2 M phosphate buffer (pH 7.98)/6 M
guanidine.HCl for a final concentration of .about.1 mM. The mixture
was irradiated with UV at 365 nm, 28.degree. C. for 2 hours.
Subsequently, 20% MPA was added and the reaction was left at
37.degree. C. for 12 hours. After completion of thioester formation
the Ub-SR was purified using preparative RP-HPLC on C4 column and a
linear gradient of 10-60% B over 30 minutes. The fractions were
analyzed by ESI-MS and the desired fractions were collected,
lyophilized to afford ubiquitin thioester 5 in 30% yield.
EXAMPLE 5
[0280] Obiqultylatlon by the Synthesized Ub-SR (5) on
.alpha.-synuclein(1-17) Model Peptide:
[0281] This example corresponds to Scheme 6.
[0282] Ub-SR was tested in peptide ubiquitylation using
.alpha.-synuclein (1-17) model peptide, as detailed below:
[0283] The .alpha.-synuclein (1-17) model peptide (7) was prepared
using Boc solid phase peptide synthesis.
Ligation of Peptide (7) with Ubiquitin Thioester (5):
[0284] Purified peptides 5, (1.60 mgrams, 1 eq) and 7 (1 mgrams, 3
eq) were dissolved in 100 .mu.L of 6 M guanidine. HCl, 200 mM
phosphate buffer pH 7.98 (Due to TFA salts, after cleavage the
actual pH after mixing the peptide was .about.7.0). To this
solution 2 .mu.L each of benzyl mercaptan and thiophenol were added
and incubated for 5 hours at 37.degree. C. The reaction was
followed using analytical column and a gradient of 10-60% B over 30
minutes. For preparative HPLC a similar gradient was used to afford
the ligation product 8 (according to numbering in Scheme 6) in -60%
yield (-1.0 mg).
[0285] Desulfurization: The ubiquitylated peptide was dissolved in
argon purged 6 M guanidine.HCl 0.2 M Phosphate buffer pH 7.98 to a
concentration of 2 mM. To this solution, a 0.5 M solution of TCEP
in argon purged guanidine.HCl phosphate buffer pH 7.98 and 10%
(v/v) of t-BuSH and 0.1 M radical initiatior VA-044 were added,
sequentially. The mixture was left at 37.degree. C. for 3 hours.
The extent of reaction was analyzed using C-4 analytical RP-HPLC
employing a gradient of 10-60% B over 30 minutes to yield 75% of
pure desulfurized peptide 8.
Enzymatic Cleavage of Isopeptide
[0286] Purified peptide 8 was dissolved in 482 .mu.L of assay
buffer (50 mM Tris, 150 mM NaCl, 1 mM DTT, pH 7.5) to a final
concentratio of .about.100 .mu.M and reacted with recombinant human
ubiquitin C-terminal hydrolase L3 (UCH-L3, Aldrich). 10 .mu.g of
UCH-L3 in 15.5 .mu.L of assay buffer containing 50 mM Tris, 150 mM
NaCl, 12 mM DTT, pH 8.0 was incubated for 20 minutes at 25.degree.
C. To the reduced UCH-L3 was then added 8 in 187 .mu.L. The mixture
was incubated for 12 hours at 37.degree. C., at which a complete
hydrolysis was achieved. The reaction was analyzed using C-4
analytical RP-HPLC employing a gradient of 10-60% B for 30 minutes,
in order to identify the hydrolysis.
EXAMPLE 6
[0287] Preparation of Thz-protected mercaptolysine:
[0288] Thz-protected mercaptolysine was prepared by the following
steps. The compound numbers correspond to the numbers on Schemes
7-10:
Preparation of protected amino acid 3b:
[0289] This example corresponds to Scheme 8.
[0290] A 50 mL round-bottom flask equipped with argon inlet, a
rubber septum, and a stirring bar, was charged with trityl thiol
(2.19 mmol) in dry THF (10 mL), and cooled to -40.degree. C. To
this solution was added NaHMDS (0.6 M, 3.66 mL, 2.19 mmol), and
stirred for 10 minutes. The reaction mixture was cooled to
-78.degree. C. and stirring was continued for additional 5 minutes.
A solution of nitro olefin, 2 (1.83 mmol) in dry THF (15 mL) was
then added over a period of 10 minutes. After 40 minutes of
stirring, the reaction was quenched with saturated aqueous solution
of NH.sub.4Cl (5 mL) , diluted with water (20 mL) and extracted
with ethyl acetate (3.times.15 mL). The combined organic extracts
were washed with brine (15 mL), dried over Na.sub.2SO.sub.4 and
purified using flash column chromatography (silica gel, ethyl
acetate/n-hexane) afforded .about.1:1 diastereomeric mixture of
Compound 3b in Yield: 1.08 grams (89%). Rf=0.51 (ethyl
acetate/n-hexane 1/4)
Analysis of Compound 3:
[0291] .sup.1H NMR (500 MHz, CDCl.sub.3, 20.degree. C., TMS) : d
1.40 (s, 9H; --OC(CH.sub.3).sub.3), 1.41 (9H;
--OC(CH.sub.3).sub.3), 1.53-1.69 (m, 2H; H-2), 1.79-1.87,
2.05-2.09, 2.17-2.24 (m, 2H; H-3) , 2.92-2.96 (m, 1H; H-4), 3.10
and 3.17 (2 x dd, J=3.9, 13.1 Hz, 1H; H-5a), 3.60, 3.63 (2 x s, 3H;
--OCH.sub.3), 3.87 and 3.94 (2 x dd, J=10.7, 13.3 Hz, 1H; H-5b) ,
4.66-4.71 (m, 1H; H-1), 7.14-7.17 (m, 3H; ArH), 7.20-7.24 (m, 6H;
ArH), 7.42-7.46 (m, 6H; ArH); .sup.13C NMR (125 MHz, CDC13,
20.degree. C.) : d 25.9 (C-2); 26.0 (2 x --OC(CH.sub.3).sub.3),
28.8 (C-3), 29.0 (C-4), 41.4 (--SC(Ph).sub.3), 52.2 (OCH.sub.3),
57.6 (C-1), 77.4 (C-5), 83.3 (2 x --OC(CH.sub.3).sub.3), 127.0 (3 x
ArCH) , 128.2 (6 x ArCH) , 129.2 (6 x ArCH), 144.1 (3 x ArC), 151.8
(2 x --NC (O)OC(CH.sub.3).sub.3), 170.8 (--C (O)OCH.sub.3).
Preparation of Intermediate Nitro Compound 4 by Replacing the
Di-Boc in the Protected Amino Acid 3 to Fmoc Protection:
[0292] This example corresponds to Scheme 9.
[0293] The protected amino acid 3b (1.08 grams, 1.62 mmol) was
dissolved in dry ethyl acetate (25 mL) and cooled to -20.degree. C.
followed by purging with dry HCl (g). After 1 hour, the mixture was
concentrated and dried to give the hydrochloride salt of the
corresponding amine. The resulting amine hydrochloride was
dissolved in Dioxane-Water (2:1) (6 mL) and aqueous sodium
bicarbonate solution (4M, 6.47 mmol) was added, stirred for 5
minutes at RT. Fmoc-Osu (0.55 grams, 1.63 mmol) was dissolved in
dioxane (3.5 mL) and added dropwise to the previous mixture. After
2 hours of stirring, the reaction mixture was poured into water and
extracted using ethyl acetate (3.times.15 mL). The combined organic
layers were dried (Na.sub.2SO.sub.4), concentrated and purified
using flash column chromatography (silica gel, ethyl
acetate/n-hexane 25/75) afforded 4 as a thick liquid (0.72 grams,
96% over two steps): R.sub.f=0.56 (ethyl acetate/n-hexane 3/7).
Analysis of compound 4:
[0294] .sup.3H NMR (500 MHz, CDCl.sub.3, 20.degree. C., TMS):
.delta.1.47-1.72 (m, 2H; H-2), 1.77-1.84, 1.91-2.05 (m, 2H; H-3) ,
2.81-2.88 (m, 1H; H-4), 3.26 and 3.45 (2 x dd, J=3.8, 13.1 and 3.9,
13.1 Hz, 1H; H-5a), 3.62 (s, 3H; OCH.sub.3), 3.86-3.93 (m, 1H;
H-5b), 4.11 (t, J=6.8 Hz, 1H; CH(Fmoc)), 4.20-4.23 (m, 1H; H-1),
4.27-4.30 (m, 2H; CH.sub.2(Fmoc)), 5.15-5.18 (m, 1H; NH), 7.09-7.13
(m, 3H; ArH) , 7.16-7.27 (m, 8H; Fmoc), 7.25-7.32 (m, 2H; ArH),
7.41 (d, J=7.8 Hz, 6H; ArH), 7.49 (t, J=6.3 Hz, 2H; ArH), 7.63-7.67
(m, 2H; ArC);
[0295] .sup.13C NMR (125 MHz, CDCl.sub.3, 20.degree. C. ):
.delta.27.9 (C-2), 28.3 (C-3), 28.7 (--C(Ph).sub.3), 41.2 (C-4),
47.0 (CH(Fmoc)), 52.4 (OCH.sub.3), 53.4 (C-1), 67.0
(CH.sub.2(Fmoc)), 77.3 (C-5), 119.9 (2 x ArCH), 125.0 (2 x ArCH),
127.0 (3 x ArCH), 127.6 (2 x ArCH), 128.2 (6 x ArCH), 129.2 (6 x
ArCH), 141.2 (ArC), 143.7 (ArC), 143.9 (3 x ArC), 155.7 (--NHC (O)
O (Fmoc)), 172.2 (--C(O)OCH.sub.3).
[0296] ESI-MS: Calculated for
[C.sub.41H.sub.38N.sub.2O.sub.6S.Na.sup.+].sup.+: 709.8 Da,
Observed: 709.3 Da.
Preparation of Intermediate Compound 5 by Reduction of the Nitro
Group, Deprotection of the Trityl Group, Incorporation of
Thiazolidine, and N-Boc Protection of Intermediate Nitro Compound
4:
[0297] This example corresponds to Scheme 9.
[0298] To a stirred solution of nitro compound 4 (0.24 grams, 0.36
mmol) in methanol (14 mL) at room temperature was added Zn poweder
(0.47 grams, 7.11 mmol) followed by dropwise addition of 10% HCl
solution (2.5 mL). The reaction was stirred at room temperature
until starting material was consumed based on TLC. The reaction
mixture was filtered through celite-545 bed, concentrated, and
dried on high vacuum. The dried amine hydro chloride was treated
with TFA-CH.sub.2Cl.sub.2 (1:1) (4 mL) and TIS (0.11 mL, 0.53 mmol)
for 1 hour at room temperature, then concentrated, dried under high
vaccum. The crude product was dissolved in eOH-water (4:1) (5 mL) ,
followed by the addition of sodium bicarbonate (0.03 grams, 0.36
mmol) and the mixture was stirred for 15 minutes. To this mixture,
formaldehyde (37% in water) (0.03 mL, 0.39 mmol) was added and
stirred for 15 hours at 25.degree. C. Subsequently, (Boc).sub.2O
(0.09 grams, 0.43 mmol) was added and stirring was continued for
additional 15 hours. The reaction mixture was concentrated and
extracted using ethyl acetate (3.times.15 mL). The combined organic
layers were dried, concentrated, and purified using flash column
chromatography (silica gel, ethyl acetate/n-hexane 25/75) to afford
5 as a foamy white solid (0.076 grams, 40% over four steps).
Analysis of compound 4:
[0299] .sup.1H NMR (500 MHz, CDCl.sub.3, 20.degree. C., TMS):
.delta.1.47-1.48 (2 x s, 9H; OC(CH.sub.3).sub.3), 1.60-1.80 (m, 3H;
H-2, H-3a), 1.92-2.06 (m, 1H; H-3b), 3.39 (m, 2H; H-4, H-5a),
3.73-3.76 (m, 1H; H-5b), 3.74 (s, 3H; COOCH.sub.3), 4.23 (t, J=6.9
Hz, 1H; CH(Fmoc)), 4.39-4.43 (m, 5H; H-1, H-6, CH.sub.2(Fmoc)),
5.37 (d, J=8.2 Hz, 1H; NH), 7.30-7.35 (m, 2H; ArH), 7.39-7.43 (m,
2H; ArH), 7.59-7.61 (m, 2H; ArH), 7.77-7.78 (m, 2H; ArH);
[0300] .sup.13C NMR (125 MHz, CDCl.sub.3, 20.degree. C.):
.delta.28.3 (--C(CH.sub.3).sub.3), 29.6 (C-2), 31.4 (C-3), 46.1
(C-4), 47.1 (CH-Fmoc), 48.0 (C-5), 52.5 (OMe), 53.7 (C-1), 54.0
(SCH.sub.2N), 66.9 (CH.sub.2(Fmoc)), 80.5 (O--C(CH.sub.3).sub.3),
119.9 (2 x ArC), 125.0 (2 x ArC), 127.0 (2 x ArC), 127.6 (2 x ArC),
141.2 (2 x ArC), 143.6 (2 x ArC), 153.6 (--N
(CO)OC(CH.sub.3).sub.3), 155.8 (NHC (O)O (Fmoc)), 172.5
(--C(O)OMe).
[0301] ESI-MS: Calculated for
[C.sub.28H.sub.34N.sub.2O.sub.6S.Na.sup.+].sup.+: 549.6 Da,
Observed: 549.3 Da.
Preparation of Protected Mercaptolysine 1b from intermediate
compound 5:
[0302] This example corresponds to Scheme 9.
[0303] To an ice-cooled solution of 5 (0.076 grams, 0.14 mmol) in
THF-water (4:1) (2.5 mL) 0.3 M solution of LiOH (0.018 grams, 0.43
mmol) in water was added in three portions over a period of 10
minutes After stirring at 0.degree. C. for 1 hour, the pH of
reaction mixture was adjusted to pH 3-4 using cold 10% (w/v) citric
acid solution, and extracted with ethyl acetate (5.times.10 mL).
The combined organic layers were dried, concentrated, and purified
using flash column chromatography (silica gel, MeOH/CHCl.sub.3 3/7)
to give 1b as a thick mass (0.061 grams, 84%): R.sub.f=0.25
(MeOH/CHCl.sub.3 1/4).
Analysis of Compound 1b:
[0304] .sup.1H NMR (500 MHz, CDCl.sub.3, 20.degree. C., TMS):
.delta. 1.38-1.39 (2 x s, 9H; OC(CH.sub.3).sub.3), 1.51-1.72 (m,
3H; H-2, H-3a), 1.87-1.98 (m, 1H; H-3b), 3.29-3.31 (m, 2H; H-5a,
H-4), 3.57-3.67 (m, 1H; H-5b), 4.13 (t, J=6.7 Hz, 1H; CH(Fmoc)),
4.23-4.44 (m, 5H; H-1, SCH.sub.2N, CH.sub.2(Fmoc)), 5.48-5.57 (m,
1H; (exchangeable with D.sub.2O) NH), 6.01-6.21 (m, 1H;
(exchangeable with D.sub.2O), COOH), 7.19-7.24 (m, 2H; ArH), 7.30
(t, J=7.4 Hz, 2H; ArH), 7.49-7.52 (m, 2H; ArH), 7.67 (d, J=7.5 Hz,
2H; ArH);
[0305] .sup.13C NMR (125 MHz, CDCl.sub.3, 20.degree. C.) .delta.
28.3 (--C(CH.sub.3).sub.3), 29.8 (C-2), 31.1 (C-3), 45.9 (C-4),
47.0 (CH(Fmoc)), 47.9 (C-5), 53.4 (C-1), 53.9 (SCH.sub.2N), 67.0
(OCH.sub.2 (Fmoc)), 80.9 (OC(CH.sub.3).sub.3), 119.9 (2 x ArC),
125.0 (2 x ArC), 127.0 (2 x ArC), 127.7 (2 x ArC), 141.2 (2 x ArC),
143.7 (2 x ArC), 153.9 (--NC(O)OC(CH.sub.3).sub.3), 156.1
(NHC(O)O(Fmoc)), 175.4 C(O)OH).)
[0306] ESI-MS: Calculated for
[C.sub.27H.sub.32N.sub.2O.sub.6S.Na.sup.+].sup.+: 535.6. Observed:
535.3 Da.
EXAMPLE 7
[0307] Preparation of Thioester Ub Containing a 1,2 Thioamine Group
(Modified Thioesters): In one example, ubiquitin thioesters
containing THZ-protected mercaptolysine, prepared according to
Example 6, as an unnatural amino acid in position 48, instead of
the natural Lys in that position, according to the process of
Example 1. The subsequent stages (Examples 2, 3 and 4) were
repeated in the same manner to obtain the product
Ub.sub.k48-thioester in 30% overall yield. The structure was
confirmed by MS and HPLC.
[0308] In another example, the SPPS synthesis of fragment 1 (as in
Example 1) was continued in full to obtain the entire Ub
polypeptide by SPPS, with the modification that the thiol
protecting group on the LTF was trityl and further by replacing the
K amino acid at position 48 with a Thz-protected mercaptolysine, as
prepared according to Example 6. The final release from the solid
support was done using TFA/TIS/H.sub.2O (95:2.5:2.5), and was
followed by reaction with MPA at a pH ranging from 1 to 2 to obtain
the final product in a 20-30% pure yield. The structure was
confirmed by MS and HPLC.
[0309] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable
subcombination.
[0310] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims. All
publications, patents and patent applications mentioned in this
specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention.
Sequence CWU 1
1
10176PRTHomo sapiens 1Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys
Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn
Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp
Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly
Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu
His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 276PRTArtificial
SequenceSynthetic 2Leu Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr
Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val
Lys Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln
Gln Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg
Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His
Leu Val Leu Arg Leu Arg Gly Gly 65 70 75 331PRTArtificial
SequenceSynthetic 3Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser
Asp Tyr Asn Ile 1 5 10 15 Gln Lys Glu Ser Thr Leu His Leu Val Leu
Arg Leu Arg Gly Gly 20 25 30 445PRTArtificial SequenceSynthetic
4Leu Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1
5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe 35
40 45 549PRTArtificial SequenceSynthetic 5Ala Lys Ile Gln Asp Lys
Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu 1 5 10 15 Ile Phe Ala Gly
Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr 20 25 30 Asn Ile
Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly 35 40 45
Gly 627PRTArtificial SequenceSynthetic 6Leu Gln Ile Phe Val Lys Thr
Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp
Thr Ile Glu Asn Val Lys 20 25 718PRTArtificial SequenceSynthetic
7Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu 1
5 10 15 Ile Phe 831PRTArtificial SequenceSynthetic 8Cys Gly Lys Gln
Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile 1 5 10 15 Gln Lys
Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 20 25 30
949PRTArtificial SequenceSynthetic 9Cys Lys Ile Gln Asp Lys Glu Gly
Ile Pro Pro Asp Gln Gln Arg Leu 1 5 10 15 Ile Phe Ala Gly Lys Gln
Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr 20 25 30 Asn Ile Gln Lys
Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly 35 40 45 Gly
1018PRTArtificial SequenceSynthetic 10Cys Lys Ile Gln Asp Lys Glu
Gly Ile Pro Pro Asp Gln Gln Arg Leu 1 5 10 15 Ile Phe
* * * * *