U.S. patent application number 12/105912 was filed with the patent office on 2008-08-14 for purification of polypeptides.
This patent application is currently assigned to Universite de Geneve. Invention is credited to Keith Rose, Matteo Villain, Jean Vizzavona.
Application Number | 20080194872 12/105912 |
Document ID | / |
Family ID | 9890385 |
Filed Date | 2008-08-14 |
United States Patent
Application |
20080194872 |
Kind Code |
A1 |
Rose; Keith ; et
al. |
August 14, 2008 |
PURIFICATION OF POLYPEPTIDES
Abstract
This invention relates to a process for purifying a polypeptide,
a capture tag useful for purifying a polypeptide and a
periodate-cleavable amino acid derivative useful for purifying a
polypeptide.
Inventors: |
Rose; Keith; (Geneva,
CH) ; Villain; Matteo; (Geneva, CH) ;
Vizzavona; Jean; (Geneva, CH) |
Correspondence
Address: |
DARBY & DARBY P.C.
P.O. BOX 770, Church Street Station
New York
NY
10008-0770
US
|
Assignee: |
Universite de Geneve
Geneva
CH
Atheris Laboratories, Dr. Reto Stocklin et Sylvie Stocklin
associes
Plan-les-Ouates
CH
|
Family ID: |
9890385 |
Appl. No.: |
12/105912 |
Filed: |
April 18, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10258191 |
Oct 18, 2002 |
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PCT/GB01/01803 |
Apr 20, 2001 |
|
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12105912 |
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Current U.S.
Class: |
562/567 ;
562/553 |
Current CPC
Class: |
Y10S 530/812 20130101;
C07K 1/22 20130101 |
Class at
Publication: |
562/567 ;
562/553 |
International
Class: |
C07C 229/02 20060101
C07C229/02 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 2000 |
GB |
009918.4 |
Claims
1-24. (canceled)
25. An amino acid derivative selected from
T-L-CO--CR'(R'')--NH--CHR.sup.1--COOH
T-L-CR(OH)--CR'(R'')--NH--CHR.sup.1--COOH
T-L-CR(NH.sub.2)--CR'(R'')--NH--CHR.sup.1--COOH wherein T is a
capture tag group capable of binding, with or without the
participation of the --CR--(OH)--, --CR(NH.sub.2)-- or --CO--
group, with a purification matrix L is a divalent linker moiety or
may be absent R.sup.1 is a side chain of a naturally occurring
amino acid R, R' and R'' are independently selected from hydrogen,
alkyl, aralkyl, aryl and heterocyclic groups, and protected
derivatives, salts and activated derivatives thereof.
26. A protected amino acid derivative of the formula
pg.sup.1-NH--CH.sub.2--CH(Opg.sup.2)--CH.sub.2(Npg.sup.3)--CHR.sup.1CO--O-
H wherein pg.sup.1, pg.sup.2 and pg.sup.3 are the same or different
and are protecting groups compatible with solid phase peptide
synthesis, R.sup.1 is the side chain of an amino acid and salts and
activated derivatives thereof.
27. A protected amino acid derivative of the formula
pg.sup.4-Cys(pg.sup.5)-NH--CH.sub.2--CH.sub.2--CH(Opg.sup.6)-CH.sub.2
N(pg.sup.7)-CHR.sup.1CO--OH wherein pg.sup.4, pg.sup.5, pg.sup.6
and pg.sup.7 are the same or different and are protecting groups
compatible with solid phase peptide synthesis, R.sup.1 is the side
chain of an amino acid and salts and activated derivatives
thereof.
28. A kit for purification of a polypeptide, the kit comprising an
amino acid derivative according to claim 25 and a purification
matrix comprising aldehyde or ketone groups.
Description
[0001] All documents cited herein are incorporated by reference in
their entirety.
TECHNICAL FIELD OF THE INVENTION
[0002] This invention relates to a process for purifying a
polypeptide, a capture tag useful for purifying a polypeptide and a
periodate-cleavable amino acid derivative useful for purifying a
polypeptide.
BACKGROUND OF THE INVENTION
[0003] The Need for Longer Synthetic Polypeptides
[0004] With the recent advances in knowledge coming from gene
sequencing and direct protein identification projects, there is a
great need for proteins and polypeptides to study function.
Ideally, such proteins are made chemically since this approach
allows not only rapid access, but also full flexibility of
incorporation of reporter groups (fluorophores, stable isotope
labels, etc.) and other components which are not coded for
genetically. Several chemically synthesized polypeptides and
proteins are already (and others are being evaluated as potential)
in vivo diagnostic and therapeutic agents (drugs). The chemical
synthesis of polypeptides is now routine: for the solution
approach, see e.g. Sakakibara, S (1999) Biopolymers 51:279-296
"Chemical synthesis of proteins in solution", and Proc Natl Acad
Sci USA (1998) 95:13549-13554; for the solid phase approach, see
Methods in Enzymology vol. 289 "Solid phase peptide synthesis" and
e.g. Miranda L P & Alewood P F (1999) Proc Natl Acad Sci USA
96:1181-1186 "Accelerated chemical synthesis of peptides and small
proteins", and Kochendoerfer, G G & Kent, S B (1999) Curr Opin
Chem Biol 3:665-671 "Chemical protein synthesis". Nonetheless, it
is currently difficult to synthesize and purify polypeptides
greater than about 60 residues in length, so that longer
polypeptides are generally not synthesized by solid phase peptide
synthesis (SPPS). Instead, two or several shorter polypeptides are
synthesized and these are deprotected, purified, coupled together
in pairs in solution and the final product is then repurified. In
general, peptides possessing N-terminal Cys are used in a coupling
reaction referred to as ((native chemical ligation)) (Cotton, G J
& Muir T W (1999) Chemistry & Biology 6:R247-R256 "Peptide
ligation and its application to protein engineering").
[0005] Methods capable of facilitating ligation at residues other
than Cys would be most useful since they would extend the range of
polypeptides accessible to total chemical synthesis. Such methods
are being developed by various groups, and it is now possible to
ligate using an N-terminal Gly, homo-Cys (which becomes Met upon
alkylation), and His. The ability to synthesize routinely by solid
phase methodology polypeptides of 100-120 residues (i.e. entire
small proteins, or fragments for chemical ligation) will have a
major impact on the proportion of proteins coded for by the genome
which are synthetically accessible, and modular chemical synthesis
(fragments) and resin splitting permits easy access to variants and
labelled versions.
[0006] The Problem of Deletions Arising from Incomplete
Coupling
[0007] It would be useful to be able to synthesize longer
polypeptides directly by SPPS, thus avoiding the time and effort
needed to synthesize, purify and ligate several shorter fragments.
Also, if longer polypeptides could be made in good yield and purity
then even longer polypeptides could be made by ligating two or more
of such longer fragments. Unfortunately, as is well known, the
chemistry used to add amino acid residues during SPPS is not quite
quantitative, and so each cycle gives rise to an impurity (which is
the first member of a set of impurities, growing with every
succeeding cycle) which contains a deletion at that cycle. Thus,
with every cycle, there is a small loss of yield of correct
(full-length) product, and in particular there is an increase in
the complexity of the range of deletion peptides present. For
example, if the coupling reaction achieves 99% yield at each step,
after 100 such reactions there will be 0.99.sup.100.times.100%=37%
correct (full-length) product, and 63% (in molar terms) of an
astronomical number (2.sup.100=10.sup.30) of impurities lacking at
least one amino acid residue. Practically, this astronomical number
is actually limited by the Avogadro number: since most syntheses
are performed on a millimole scale, the number of impurities is
limited to about 10.sup.20. If the coupling reaction is 95%
efficient, the yield after 100 steps falls to
0.95.sup.100.times.100=0.59%, and the mixture of (theoretically)
10.sup.20-10.sup.30 impurities now accounts for 99.41% (on a moles
basis). Clearly, it is important to force coupling (and
deprotection) reactions to be as quantitative as possible in order
to obtain good yield, and it is also important to be able to purify
wanted full-length product from a myriad of impurities. This
problem of deletions arising from incomplete couplings is well
known (Methods in Enzymology, vol 289, devoted to Solid Phase
Peptide Synthesis).
[0008] Capping Reduces Complexity
[0009] Synthesis and subsequent purification of polypeptides can be
facilitated by a strategy involving <<capping>> and
<<affinity isolation>>, both of which are now
explained. By driving couplings to completion (quantitatively
<<capping>> the last trace of free amine with a high
concentration of a powerful and unhindered reagent such as acetic
anhydride), crude product complexity is reduced as the capped
chains are terminated and cannot give rise to further (exponential)
complexity through deletions during further cycles. In the final
cycle, after capping of this cycle as after every previous cycle,
the last residue to be added is to be found uniquely on full-length
material, not on the capped (truncated, terminated) chains. This
capping (with an irreversible acyl group such as acetyl) does not
increase yield, which remains at 37% for 100 steps at 99%, but it
drastically cuts the complexity of the impurity profile. If capping
achieves complete termination of deletion chains at each cycle, the
final product is contaminated with 101 impurities (the most
abundant of which is present at 1% and arose at the first cycle,
and the least abundant is present at 0.37% and arose at the b
100.sup.th cycle) instead of 10.sup.20-10.sup.30. This approach
(capping) to the problem of deletions arising from incomplete
couplings is well known (Methods in Enzymology, vol 289).
[0010] Isolation Relying on Special Properties of the
Amino-Terminal Residue
[0011] Solid phase synthesis of long polypeptides proceeds from
C-terminus (attached to the resin via a linker) to N-terminus. When
isolating the full-length polypeptide from the capped impurities
(truncated chains) present, it is better to rely on unique
properties (all or nothing) of the N-terminal residue or group than
to try a brute-force separation based on general factors such as
size, hydrophobicity, charge, etc., which do not differ greatly
between one long polypeptide and another. An approach to isolation
which relies on the properties of the N-terminal residue is also
useful when isolating recombinant DNA-derived polypeptides or
polypeptides from natural sources.
SUMMARY OF THE PRESENT INVENTION
[0012] A new approach which facilitates the purification of
polypeptides is disclosed. The approach is applicable to
polypeptides from many sources, including natural sources,
biosynthetic sources (via recombinant DNA and modified organisms),
and total chemical synthesis. It consists of three key features:
(i) polypeptides which have amino-terminal Cys, Thr or Ser are
purified by forming a covalent bond (thiazolidine or oxazolidine)
between the amino-terminal residue and a purification matrix
(generally a solid support possessing aldehyde groups), washing
away impurities and then releasing the bound peptide by reversal of
thiazolidine (or oxazolidine) formation; (ii) polypeptides which do
not have amino-terminal Cys, Thr or Ser are synthesized with an
auxiliary capture tag comprising two closely-spaced nucleophiles
(usually a vicinal-amino-thiol or a vicinal-amino-ol) attached at
or close to the amino terminal residue, directly or via a linker,
and which permits capture usually through thiazolidine or
oxazolidine formation between itself and a purification matrix
(generally a solid support possessing aldehyde groups), and after
washing away any polypeptides which do not possess the auxiliary
group, the desired full-length polypeptide is eluted from the
matrix by reversal of the covalent capture reaction; alternatively,
an auxiliary capture tag comprising an aldehyde or keto moiety is
attached at or close to the amino terminal residue, directly or via
a linker, and permits capture usually through thiazolidine or
oxazolidine formation between itself and a purification matrix
(generally a solid support possessing vicinal amino-thiol groups);
(iii) the auxiliary capture tag (comprising a capture tag group and
an associated linker) can be removed under gentle conditions,
usually through oxidation with periodate. Special procedures are
required for N-terminal Pro, and for pyroglutamyl, acetyl or other
N-blocked polypeptides. In all aspects of the present invention the
polypeptide may optionally be further purified by HPLC, RP-HPLC or
any other appropriate method.
[0013] In a first aspect, the present invention provides a process
for the purification of a polypeptide comprising a
vicinal-amino-thiol, vicinal-amino-hydroxyl, vicinal-diol, or
vicinal-diamino group the process comprising the steps of: [0014]
(i) attaching the polypeptide to a purification matrix by
contacting the polypeptide with a purification matrix comprising
aldehyde or ketone groups under conditions which favour formation
of a heterocyclic ring system, [0015] (ii) washing the purification
matrix to remove unbound material, and [0016] (iii) releasing the
polypeptide from the purification matrix.
[0017] Preferably, the polypeptide is released from the
purification matrix by exposure to conditions which reverse the
formation of the heterocyclic ring system.
[0018] Reaction of a vicinal-amino-thiol or vicinal-amino-hydroxyl
group with an aldehyde/ketone gives a five-membered thiazolidine or
oxazolidine ring, respectively. Reaction of vicinal-diol or
vicinal-diamino groups with aldehyde/ketones gives analogous
five-membered heterocyclic ring systems.
[0019] According to a preferred embodiment, the present invention
provides a process for the purification of a polypeptide comprising
an N-terminal cysteinyl (Cys) amino acid, the process comprising
the steps of [0020] (i) attaching the polypeptide to a purification
matrix by contacting the polypeptide with a purification matrix
comprising aldehyde or ketone groups under conditions favouring
formation of a thiazolidine ring, [0021] (ii) washing the
purification matrix to remove unbound material, and [0022] (iii)
releasing the polypeptide from the purification matrix by exposure
to conditions which reverse thiazolidine formation.
[0023] According to a further preferred embodiment, the present
invention provides a process for the purification of a polypeptide
comprising an N-terminal threonyl (Thr) amino acid, the process
comprising the steps of [0024] (i) attaching the polypeptide to a
purification matrix by contacting the polypeptide with a
purification matrix comprising aldehyde or ketone groups under
conditions favouring formation of a oxazolidine ring, [0025] (ii)
washing the purification matrix to remove unbound material, and
[0026] (iii) releasing the polypeptide from the purification matrix
by exposure to conditions which reverse oxazolidine formation.
[0027] According to a further preferred embodiment, the present
invention provides a process for the purification of a polypeptide
comprising an N-terminal seryl (Ser) amino acid, the process
comprising the steps of [0028] (i) attaching the polypeptide to a
purification matrix by contacting the polypeptide with a
purification matrix comprising aldehyde or ketone groups under
conditions favouring formation of a oxazolidine ring, [0029] (ii)
washing the purification matrix to remove unbound material, and
[0030] (iii) releasing the polypeptide from the purification matrix
by exposure to conditions which reverse oxazolidine formation.
[0031] According to a further aspect of the invention, the
polypeptide can be produced by recombinant DNA technology and
purification of the polypeptide may be followed by cleavage of the
N-terminal Cys, Thr or Ser amino acid residue, and optionally by
some of the further N-terminal amino acids, by enzymatic
proteolysis.
[0032] In the case of polypeptides comprising an N-terminal
cysteinyl, threonyl or seryl amino acid residue, purification of
the polypeptide can be achieved using the N-terminal
vicinal-amino-thiol or vicinal-amino-hydroxyl functionalities
present in the polypeptides.
[0033] In the case of polypeptides not possessing an N-terminal
cysteinyl, threonyl or serinyl amino acid residue, a covalent
capture tag can be employed in the purification of such
polypeptides. The capture tag is covalently bonded to the
polypeptide, preferably at or near the N-terminus of the
polypeptide. The capture tag is capable, either alone or in
combination with the polypeptide, of binding (covalently or
non-covalently) with a purification matrix. The invention further
provides a capture tag that is also cleavable from the
polypeptide.
[0034] The term "near" the N-terminus of the polypeptide means
within the first 20, preferably 10, more preferably 5, more
preferably 2 N-terminal amino acids. More preferably, the capture
tag is covalently bonded to the polypeptide at the first N-terminal
amino acid of the polypeptide and more preferably at the N-terminal
amino group of the polypeptide.
[0035] According to the present invention there is provided a
compound comprising a polypeptide covalently bonded to a capture
tag wherein the capture tag, either alone or in combination with
the polypeptide, is capable of binding to a purification matrix,
and wherein the capture tag is cleavable from the polypeptide.
[0036] The present invention further provides a process for
purifying a polypeptide comprising the the steps of
[0037] (i) preparing the polypeptide covalently bonded to a capture
tag,
[0038] (ii) attaching the polypeptide to a purification matrix by
means of the capture tag,
[0039] (iii) washing the purification matrix to remove unbound
material,
[0040] (iv) releasing the polypeptide from the matrix, and
[0041] (v) cleaving the capture tag from the polypeptide.
[0042] Preferably, the polypeptide is attached to the purification
matrix covalently, preferably by formation of a heterocyclic ring.
Preferably, the capture tag, alone or in combination with the
polypeptide, comprises a 1,2-dinucleophile wherein the nucleophiles
are selected from amino, hydroxyl and thiol, the purification
matrix comprises aldehyde or ketone groups, and the polypeptide is
attached to the matrix by formation of a five-membered heterocycle
between the dinucleophile and the aldehyde or ketone groups.
[0043] Preferably, the capture tag is cleaved from the polypeptide
by treatment with periodic acid or a salt thereof.
[0044] According to a further aspect of the present invention there
is provided a compound comprising a polypeptide and a capture tag
wherein an amino group of polypeptide and the capture tag form a
vicinal-amino-hydroxyl or vicinal-diamino group and wherein the
capture tag, either alone or in combination with said amino group
of the polypeptide, is capable of binding to a purification
matrix.
[0045] Accordingly, there is provided a process for the
purification of a polypeptide comprising a group selected from
--CR(OH)--CR'(R'')--NH--CHR.sup.1CO--
--CR(NH.sub.2)--CR'(R'')--NH--CHR.sup.1CO--
wherein [0046] R.sup.1 is the side chain of an amino acid [0047] R,
R' and R'' are selected from hydrogen, alkyl, aralkyl, aryl and
heterocyclic groups, the process comprising the steps of [0048] (i)
attaching the polypeptide to a purification matrix by contacting
the polypeptide with a purification matrix comprising aldehyde or
ketone groups under conditions favouring formation of a
thiazolidine ring, [0049] (ii) washing the purification matrix to
remove unbound material, and [0050] (iii) releasing the polypeptide
from the matrix by exposure to conditions which reverse
heterocyclic ring formation.
[0051] In this aspect of the present invention the
vicinal-amino-hydroxyl or vicinal-diamino group formed by the
capture tag and an amino group of the polypeptide is cleavable
treatment with periodic acid or a salt thereof to give the free
polypeptide.
[0052] In one embodiment of this aspect of the present invention
the polypeptide comprises the group
HOCH.sub.2--CH.sub.2--NH--CHR.sup.1.
[0053] Whilst in one aspect of the present invention there is
provided a process for purifying a polypeptide wherein the
polypeptide comprises a vicinal-amino-hydroxyl or vicinal-diamino
group which is both capable of binding to a purification matrix and
capable of cleavage by periodic acid or a salt thereof, a further
aspect of the invention provides for purification of polypeptides
comprising a capture tag group in addition to a cleavage site.
[0054] According to an embodiment of this aspect of the invention
there is provided a process for purification of a polypeptide
comprising a group
T-L-CO--CR'(R'')--NH--CHR.sup.1--CO--
wherein [0055] T is a capture tag group capable of binding, with or
without the participation of the adjacent --CO-- group, with a
purification matrix [0056] L is a divalent linker moiety or may be
absent [0057] R.sup.1 is a side chain of an amino acid [0058] R, R'
and R'' are independently selected from hydrogen, alkyl, aralkyl,
aryl and heterocyclic groups the process comprising the steps of
[0059] (i) attaching the polypeptide to a purification matrix by
contacting the polypeptide with a purification matrix comprising a
structure or chemical functionality capable of binding through the
capture tag group, [0060] (ii) washing the purification matrix to
remove unbound material, and [0061] (iii) releasing the bound
polypeptide.
[0062] According to a further embodiment of this aspect of the
invention there is provided a process for purification of a
polypeptide comprising a group
T-L-CR(OH)--CR'(R'')--NH--CHR.sup.1--CO--
wherein [0063] T is a capture tag group capable of binding, with or
without the participation of the adjacent --CR(OH)-- group, with a
purification matrix [0064] L is a divalent linker moiety or may be
absent [0065] R.sup.1 is a side chain of an amino acid [0066] R, R'
and R'' are independently selected from hydrogen, alkyl, aralkyl,
aryl and heterocyclic groups the process comprising the steps of
[0067] (i) attaching the polypeptide to a purification matrix by
contacting the polypeptide with a purification matrix comprising a
structure or chemical functionality capable of binding through the
capture tag group, [0068] (ii) washing the purification matrix to
remove unbound material, and [0069] (iii) releasing the bound
polypeptide.
[0070] According to a further embodiment of this aspect of the
invention there is provided a process for purification of a
polypeptide comprising a group
T-L-CR(NH.sub.2)--CR'(R'')--NH--CHR.sup.1--CO--
wherein [0071] T is a capture tag group capable of binding, with or
without the participation of the adjacent --CR(NH.sub.2)-- group,
with a purification matrix [0072] L is a divalent linker moiety or
may be absent [0073] R.sup.1 is a side chain of an amino acid
[0074] R, R' and R'' are independently selected from hydrogen,
alkyl, aralkyl, aryl and heterocyclic groups the process comprising
the steps of [0075] (i) attaching the polypeptide to a purification
matrix by contacting the polypeptide with a purification matrix
comprising a structure or chemical functionality capable of binding
through the capture tag group, [0076] (ii) washing the purification
matrix to remove unbound material, and [0077] (iii) releasing the
bound polypeptide.
[0078] This aspect of the invention may further comprise the step
of cleaving the group comprising the step of cleaving the group
comprising T-L-CO--, T-L-CR(OH)-- or T-L-CR(NH.sub.2)-- from the
polypeptide with periodic acid or a salt thereof.
[0079] Cleaving the group T-L-CO--, T-L-CR(OH)-- or
T-L-CR(NH.sub.2)-- from the polypeptide may serve to release the
polypeptide from the purification matrix. Alternatively, cleavage
of the group T-L-CO--, T-L-CR(OH)-- or T-L-CR(NH.sub.2)-- from the
polypeptide may be performed subsequent to releasing the bound
polypeptide from the purification matrix.
[0080] The linker L may comprise any suitable divalent spacer.
Suitable linkers include an alkylene group.
[0081] According to a further aspect of the present invention there
is provide a polypeptide comprising a group
--CR(NH.sub.2)--CR'(R'')--NH--CHR.sup.1CO--
wherein [0082] R.sup.1 is the side chain of an amino acid, [0083]
R, R' and R'' are independently selected from hydrogen, alkyl,
aralkyl, aryl and heterocyclic groups and protected derivatives
thereof.
[0084] According to a further aspect of the present invention there
is provide a polypeptide comprising a group
--CO--CR'(R'')--NH--CHR.sup.1CO--
wherein [0085] R.sup.1 is the side chain of an amino acid, [0086]
R, R' and R'' are independently selected from hydrogen, alkyl,
aralkyl, aryl and heterocyclic groups and protected derivatives
thereof
[0087] According to a further aspect of the present invention there
is provided a polypeptide comprising a group
T-L-CR(OH)--CR'(R'')--NH--CHR'--CO-- [0088] wherein [0089] T is a
capture tag group capable of binding, with or without the
participation of the --CR--(OH)-- group, with a purification matrix
[0090] L is a divalent linker moiety or may be absent [0091]
R.sup.1 is the side chain of an amino acid of the polypeptide R, R'
and R'' are independently selected from hydrogen, alkyl, aralkyl,
aryl and heterocyclic groups and protected derivatives thereof.
[0092] According to a further aspect of the present invention there
is provided a polypeptide comprising a group
T-L-CR(NH.sub.2)--CR'(R'')--NH--CHR.sup.1--CO--
wherein [0093] T is a capture tag group capable of binding, with or
without the participation of the --CR--(NH.sub.2)-- group, with a
purification matrix [0094] L is a divalent linker moiety or may be
absent [0095] R.sup.1 is the side chain of an amino acid of the
polypeptide R, R' and R'' are independently selected from hydrogen,
alkyl, aralkyl, aryl and heterocyclic groups and protected
derivatives thereof.
[0096] According to a further aspect of the present invention there
is provided a polypeptide comprising a group
T-L-CO--CR.sup.1(R'')--NH--CHR.sup.1--CO-- [0097] wherein [0098] T
is a capture tag group capable of binding, with or without the
participation of the --CO-- group, with a purification matrix
[0099] L is a divalent linker moiety or may be absent [0100]
R.sup.1 is the side chain of an amino acid of the polypeptide R, R'
and R'' are independently selected from hydrogen, alkyl, aralkyl,
aryl and heterocyclic groups and protected derivatives thereof.
[0101] In one embodiment of this aspect of the invention, the
capture tag group T is capable of binding with a purification
matrix with the participation of the --CR(OH)--, --CR(NH.sub.2)--
or --CO-- group. Preferred embodiments include polypeptides
comprising a group selected from
HS--CH.sub.2--CH.sub.2--CH(OH)CH.sub.2NH--CHR.sup.1CO--
H.sub.2N--CH.sub.2--CH.sub.2--CH(OH)CH.sub.2NH--CHR.sup.1CO--
H.sub.2N--CH.sub.2--CH(OH)CH.sub.2NH--CHR.sup.1CO--.
[0102] In an alternative embodiment, the capture tag group T is
capable of binding a purification matrix without the participation
of the --CR(OH)--, --CR(NH.sub.2)-- or --CO-- group. Preferred
embodiments include polypeptides comprising a group selected
from
H-Cys-NH--CH.sub.2--CH.sub.2--CH(OH)--CHNH--CHR.sup.1CO--
H-Thr-NH--CH.sub.2--CH.sub.2--CH(OH)--CHNH--CHR.sup.1CO--
H.sub.2N--O--CH.sub.2--CO--NH--CH.sub.2--CH.sub.2--CH(OH)--CH.sub.2NH--C-
HR.sup.1CO--.
[0103] According to a further aspect of the present invention there
is provided a process for removing the capture tag group T and,
when present, the linker L from a polypeptide comprising a group
selected from
T-L-CR(OH)--CR'(R'')--NH--CHR.sup.1--CO--
T-L-CR(NH.sub.2)--CR'(R'')--NH--CHR.sup.1--CO--
T-L-CO--CR'(R'')--NH--CHR.sup.1--CO--
the process comprising the step of treating the polypeptide with
periodic acid or a salt thereof.
[0104] According to one embodiment of this aspect of the invention,
the polypeptide is treated in solution. According to an alternative
embodiment of this aspect of the invention, the polypeptide is
treated while bound to a matrix and treatment serves to release the
polypeptide from the matrix.
[0105] According to a further aspect of the present invention there
are provided amino acid derivatives comprising cleavable capture
tags according to the invention. Such derivatives are useful in the
preparation of polypeptides according to the invention.
Accordingly, the invention provides an amino acid derivative
selected from
T-L-CO--CR'(R'')--NH--CHR.sup.1--COOH
T-L-CR(OH)--CR'(R'')--NH--CHR.sup.1--COOH
T-L-CR(NH.sub.2)--CR'(R'')--NH--CHR.sup.1--COOH
wherein [0106] T, L, R, R', R'' and R.sup.1 as is previously
defined and protected derivatives, salts and activated derivatives
thereof.
[0107] Protecting groups, particularly for the amino and hydroxyl
functions are typically required for use in polypeptide synthesis.
Preferred protecting groups are compatible with solid phase
polypeptide synthesis, linkage to the solid phase and side-chain
protection used in peptide synthesis. Examples of protecting groups
include fluorenyl-methyl-oxycarbonyl, tert-butyloxycarbonyl and
benzyloxycarbonyl.
[0108] Activated derivatives of the carboxyl group may be required
by polypeptide synthesis. Activated derivatives include alkali
metal salts, and acyl chloride, acyl fluoride, acyl imidazole,
mixed anhydride, symmetrical anhydride and active ester derivatives
of the carboxyl group.
[0109] A preferred embodiment of an amino acid derivative of the
present invention comprises a protected amino acid derivative of
the formula
pg'-NH--CH.sub.2--CH(Opg.sup.2)-CH.sub.2(Npg.sup.3)-CHR.sup.1CO--OH
wherein [0110] pg.sup.1, pg.sup.2 and pg.sup.3 are the same or
different and are protecting groups compatible with solid phase
peptide synthesis, [0111] R.sup.1 is the side chain of an amino
acid and salts and activated derivatives thereof.
[0112] Suitable protecting groups may be selected for pg.sup.1
(such as fluorenyl-methyl-oxycarbonyl or tert-butyloxycarbonyl),
pg.sup.2 and pg.sup.3 (such as fluorenyl-methyl-oxycarbonyl,
tert-butyloxycarbonyl or benzyloxycarbonyl).
[0113] A further preferred embodiment of an amino acid derivative
of the present invention comprises a protected amino acid
derivative of the formula
pg.sup.4-Cys(pg.sup.5)-NH--CH.sub.2--CH.sub.2--CH(Opg.sup.6)-CH.sub.2N(p-
g.sup.7)-CHR.sup.1CO--OH
wherein [0114] pg.sup.4, pg.sup.5, pg.sup.6 and pg.sup.7 are the
same or different and are protecting groups compatible with solid
phase peptide synthesis, [0115] R.sup.1 is the side chain of an
amino acid and salts and activated derivatives thereof
[0116] Suitable protecting groups may be selected for pg.sup.4,
pg.sup.5, pg.sup.6 and pg.sup.7 (such as
fluorenyl-methyl-oxycarbonyl or tert-butyloxycarbonyl).
[0117] According to a further aspect of the present invention there
is provided a kit for purification of a polypeptide, the kit
comprising an amino acid derivative according to the previous
aspect of the invention and a purification matrix comprising
aldehyde or ketone groups.
[0118] In all aspects of the present invention, R, R' and R'' are
independently preferably hydrogen. R.sup.1 is preferably the side
chain of a naturally occurring amino acid. R.sup.1 is preferably
the side chain of the N-terminal amino acid of the polypeptide.
[0119] According to a further aspect of the present invention there
is provided a purification matrix comprising aldehyde or ketone
groups, a process for the preparation of such a matrix and use of
such a matrix in the purification of polypeptides. Preferably, the
purification matrix comprises aldehyde groups. Suitable matrices
include amino PEGA resins derivatised to include aldehyde groups,
and matrices based on a cross-linked dextran support such as an
acetaldehyde Sephadex resin, which is compatible with solutions
containing 6M guanidinium chloride and is particularly suited to
handling inclusion bodies produced by recombinant DNA
techniques.
[0120] As used herein, the term "purification matrix" includes any
suitable solid support, gel or resin.
[0121] As used herein, the term "polypeptide" includes proteins,
oligopeptides and peptoids. The polypeptides may be obtained by any
suitable method including isolation from natural sources,
recombinant DNA technology, chemical synthesis and enzymatic
synthesis. The polypeptides may be naturally occurring or synthetic
and may be modified or derivatised chemically or enzymatically.
Typically, a polypeptide will comprise between 2 and 1000,
preferably between 2 and 500, more preferably between 2 and 500,
more preferably between 2 and 100, more preferably between 5 and
50, amino acids. Each amino acid may be a naturally occurring or
synthetic amino acid. Preferably each amino acid is a naturally
occurring amino acid such as alanine, arginine, asparagine,
aspartic acid, cysteine, cystine, glycine, glutamic acid,
glutamine, histidine, isoleucine, leucine, lysine, methionine,
phenylalanine, proline, serine, threonine, tryptophan, tyrosine,
and valine.
[0122] As used herein, the term "alkyl" means an optionally
substituted branched or unbranched, cyclic or acyclic, saturated or
unsaturated (i.e. alkenyl or alkynyl) hydrocarbyl radical. Where
acyclic, the alkyl group is preferably a C.sub.1-12, more
preferably C.sub.1-4 chain. Cyclic alkyl groups include alkyl
groups which comprise both acyclic groups (eg methyl, ethyl, propyl
etc.) and cyclic groups (eg cyclopentyl, cyclohexyl, cycloheptyl,
etc.) as well as only cyclic groups. Where cyclic, the alkyl group
is preferably a C.sub.3-12, more preferably C.sub.5-10 and more
preferably comprises a C.sub.5, C.sub.6 or C.sub.7 ring. The alkyl
ring or chain may optionally include (i.e. be interrupted by and/or
terminate with) one or more heteroatoms such as oxygen, sulphur or
nitrogen.
[0123] As used herein, the term "aryl" means an optionally
substituted C.sub.3-12 aromatic group, such as phenyl or naphthyl,
or a heteroaromatic group containing one or more, preferably one or
two, heteroatom(s), such as pyridyl, pyrrolyl, furanyl, thienyl,
pyrimidinyl, imidazolyl, triazolyl, thiazolyl, indolyl, indazolyl,
quinolinyl, isoquinolinyl, benzodiazolyl, benzotriazilyl,
benzofuranyl, benzothienyl, quinoxalinyl.
[0124] As used herein, the term "aralkyl" means an optionally
substituted branched or unbranched cyclic or acylic C.sub.4-18
group comprising an alkyl group and an aryl group (for example,
benzyl). An aralkyl group may be bonded via the alkyl or aryl
group.
[0125] As used herein, the term "alkylene" means an optionally
substituted branched or unbranched, cyclic or acyclic, saturated or
unsaturated (i.e. alkenylene or alkynylene) divalent hydrocarbyl
radical. Where acyclic, the alkylene group is preferably a
C.sub.1-12, more preferably C.sub.1-4 chain. Cyclic alkylene groups
include alkylene groups which comprise both acyclic groups (eg
methylene, ethylene, propylene etc.) and cyclic groups (eg
cyclopentylene, cyclohexylene, cycloheptylene, etc.) as well as
only cyclic groups. Where cyclic, the alkylene group is preferably
a C.sub.3-12, more preferably C.sub.5-10 and more preferably
comprises a C.sub.5, C.sub.6 or C.sub.7 ring. The alkylene ring or
chain may optionally include (i.e. be interrupted by and/or
terminate with) one or more heteroatoms such as oxygen, sulphur or
nitrogen.
[0126] As used herein, the term "heterocyclic group" means a cyclic
group containing one or more, preferably one, heteroatom (e.g.
thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl,
isothiazolyl, oxazolyl, oxadiazolyl, thiadiazolyl, pyrrolidinyl,
pyrrolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl,
tetrahydrofuranyl, pyranyl, pyronyl, pyridyl, pyrazinyl,
pyridazinyl, piperidyl, piperazinyl, morpholinyl, thianaphthyl,
benzofiranyl, isobenzofuranyl, indolyl, oxyindolyl, isoindolyl,
indazolyl, indolinyl, 7-azaindolyl, benzopyranyl, coumarinyl,
isocoumarinyl, quinolinyl, isoquinolinyl, naphthridinyl,
cinnolinyl, quinazolinyl, pyridopyridyl, benzoxazinyl,
quinoxalinyl, chromenyl, chromanyl, isochromanyl, phthalazinyl and
carbolinyl
[0127] The alkyl, aryl and aralkyl groups may be substituted or
unsubstituted. Where substituted, there are preferably one to three
substituents, more preferably one substituent. Substituents may
include halogen atoms and halogen containing groups such as
haloalkyl (e.g. trifluoromethyl); oxygen containing groups such as
alcohols (e.g. hydroxy, hydroxyalkyl, aryl(hydroxy)alkyl), ethers
(e.g. alkoxy, alkoxyalkyl, aryloxyalkyl), aldehydes (e.g.
carboxaldehyde), ketones (e.g. alkylcarbonyl, alkylcarbonylalkyl,
arylcarbonyl, arylalkylcarbonyl, arylcarbonylalkyl), acids (e.g.
carboxy, carboxyalkyl), acid derivatives such as esters (e.g.
alkoxycarbonyl, alkoxycarbonylalkyl, alkycarbonylyoxy,
alkycarbonylyoxyalkyl) and amides (e.g. aminocarbonyl, mono- or
dialkylaminocarbonyl, aminocarbonylalkyl, mono- or
dialkylaminocarbonylalkyl, arylaminocarbonyl); and carbamates (e.g.
alkoxycarbonylamino, aryloxycarbonylamino, aminocarbonyloxy, mono-
or dialkylaminocarbonyloxy, arylaminocarbonyloxy), and ureas (e.g.
mono- or dialkylaminocarbonylamino or arylaminocarbonylamino);
nitrogen containing groups such as amines (e.g. amino, mono- or
dialkylamino, aminoalkyl, mono- or dialkylaminoalkyl), azides,
nitriles (e.g. cyano, cyanoalkyl), nitro; sulfur containing groups
such as thiols, thioethers, sulfoxides, and sulfones (e.g.
alkylthio, alkylsulfinyl, alkylsulfonyl, alkylthioalkyl,
alkylsulfinylalkyl, alkylsulfonylalkyl, arylthio, arylsulfinyl,
arylsulfonyl, arylthioalkyl, arylsulfinylalkyl, arylsulfonylalkyl);
and heterocyclic groups. As used herein, the term "halogen" means a
fluorine, chlorine, bromine or iodine radical, preferably a
fluorine or chlorine radical.
BRIEF DESCRIPTION OF THE FIGURE
[0128] FIG. 1 shows HPLC chromatograns (time is left to right,
vertical axis records absorbance at 214 nm). The left panel of FIG.
1 shows a mixture of model peptides CYAKYAKL (C) and YAKYAKL (Y).
The middle panel of FIG. 1 shows the supernatant after covalent
capture of C on the aliphatic aldehyde resin after 1 h, while the
control peptide without N-terminal Cys (Y) did not bind. The right
panel of FIG. 1 shows the supernatant after 4 wash cycles which
removed any traces of unbound Y, followed by elution of C
uncontaminated with Y.
DETAILED DESCRIPTION OF THE INVENTION
[0129] Polypeptides Possessing N-Terminal Cys
[0130] Polypeptides possessing N-terminal Cys are particularly
important since they are used in native chemical ligation to make
longer polypeptides. It is well known that polypeptides possessing
N-terminal Cys are able to form a heterocyclic ring known as a
thiazolidine. This reaction has been exploited to attach a variety
of groups to the N-terminus of such polypeptides (e.g. Zhang &
Tarn 1996 Analyt. Biochem. 233:87-93). Since the reaction occurs
under mild, aqueous or semi-aqueous conditions, and is reversible,
it should in principle be possible to exploit it to bind a
polypeptide in a covalent capture step to a solid support
possessing appropriate aldehyde or ketone functions, and then to
release the polypeptide after having washed away unbound impurities
(including especially very similar polypeptides which do not
possess the N-terminal Cys capture group). Of course, peptides
possessing N-terminal Thr or Ser are in principle capable of
interacting covalently with an aldehyde column through oxazolidine
formation, and being released along with N-terminal Cys peptides in
the release step (see below). In the method which we describe,
which involves capping during synthesis, there should be no
N-terminal Thr or Ser peptides present in a preparation of an
N-terminal Cys polypeptide. We have reduced this to practice and
demonstrated the practicality of such an approach.
[0131] For example, we have succeeded in exploiting thiazolidine
formation on an amino PEGA resin (NovaBiochem, Switzerland) which
we modified with O.dbd.CH--CH.sub.2NH--COCH.sub.2CH.sub.2CO-- in
one set of experiments and with O.dbd.CH--C.sub.6H.sub.4--CO-- in
another set of experiments. This PEGA resin swells in mixed
aqueous/organic solvents and allows penetration by large
biomolecules (to at least approximately 35 kDa). It has
approximately 30 mM functional groups. Typical laboratory scale
synthesis of a long polypeptide yields a maximum of 0.1-0.2 mmoles
full-length polypeptide for purification, as a solution (post
cleavage-deprotection) in 100 ml 50% acetonitrile, which represents
a concentration of 1-2 mM, so the purification resin substitution
is more than adequate. Tris(carboxyethyl)phosphine (TCEP) but not
dithiothreitol (DTT) is compatible with thiazolidine formation and
helps prevent disulfide bond formation between a bound full-length
polypeptide and a truncated impurity. After washing away material,
which has not been covalently captured, elution is achieved under
conditions known to reverse thiazolidine formation. Such conditions
include aqueous acid, with or without additives which react with
the aldehyde function, e.g. 1% trifluoroacetic acid (TFA) in 50%
acetonitrile which is also 0.1 M in dithiothreitol (DTT); or with
0.1 M aminiooxyacetic acid hemihydrochloride in 50% acetonitrile,
with or without addition of DTT. DTT keeps thiols reduced, which is
useful to avoid mixed disulfide formation between captured
full-length peptide and otherwise free truncated material with
internal Cys. Release with DTT alone is much slower than with
aminooxy compounds, so it is possible to include DTT in a wash step
prior to elution with an aminooxy compound, or to include it with
the aminooxy release compound. Aminooxyacetic acid, commercially
available as its hemihydrochloride, is convenient, but other
compounds possessing an aminooxy group, such as methoxamine, are
suitable provided the pH is adjusted to between 1 and 7, more
preferably between 1.5 and 5, and most preferably between 2 and 3.
After release of the peptide, it is generally not worth trying to
regenerate the purification resin, which costs about 50 $ for 5 ml.
In Example 1, resin loading was rapid (1 h), release from the resin
was achieved within 24 h, and yields were good. We were also able
to elute with cysteamine (which competes by forming a thiazolidine
and helps to keep the peptide in reduced, i.e. thiol, form,
although this was not generally a problem at pH 4.5). By modulating
the structure of the aldehyde or ketone it is possible to vary
capture and release kinetics and thermodynamics. To avoid potential
reversed-phase properties of the resin which might become manifest
with very hydrophobic polypeptides, it is possible to use other
resins, such as those based on dextran or agarose, and others, and
to replace part or all of the acetonitrile (or other solubilizing
organic solvent) with guanidine hydrochloride. As is well known,
aldehyde groups are conveniently introduced into sugar-based
supports such as dextran (and modified dextran, such as Sephadex)
by oxidation with periodate, or through chemical modification of
carboxy modified or amino modified supports. For solid phase
chemical ligation (see Kochendoerfer & Kent review), N-terminal
Cys peptides are required which also possess a C-terminal
thioester: the N-terminus needs to be protected to prevent
cyclization and so can no longer be used as a purification handle.
Such cases require a non-Cys capture tag (as discussed below) or
are made as thioacids for the N.fwdarw.C solid phase ligation
approach (Canne et al. 1999 J. Am. Chem. Soc. 121:8720-8727). Of
course, peptides possessing N-terminal Cys made by recombinant DNA
techniques or found in nature, can benefit from purification by
covalent capture.
[0132] Polypeptides Possessing N-terminal Thr or Ser
[0133] It is also well known that polypeptides possessing
N-terminal Thr or Ser are able to form a heterocyclic ring known as
an oxazolidine (e.g. Tam et al. 1995 Int. J. Peptide Protein Res.
45:209-216). Since the reaction occurs under mild, aqueous or
semi-aqueous conditions, and is in principle reversible, it should
in principle be possible to exploit it to bind a polypeptide in a
covalent capture step to a solid support possessing appropriate
aldehyde or ketone functions, and then to release the polypeptide
after having washed away unbound impurities (including especially
very similar polypeptides which do not possess the N-terminal Thr
or Ser capture group). Of course, peptides possessing N-terminal
Cys are in principle capable of reacting with an aldehyde support
or column through thiazolidine formation, and being released along
with N-terminal Thr or Ser peptides in the release step (see
above). In the method which we describe, which involves capping
during synthesis, there should be no N-terminal Cys peptides
present in a preparation of an N-terminal Thr or Ser polypeptide.
Tam et al. Int. J. Peptide Protein Res. 45 (1995) 209-216 give
details of oxazolidine formation with N-terminal Thr and Ser: Thr
reacts in aqueous solution pH 7 with t.sub.1/2>300 h, but
rapidly and completely within 20 h when organic co-solvent is
present. Tarn reports that Ser reacts slowly and incompletely (25%)
even when co-solvent is present. Depending on the structure of the
aldehyde or ketone, the kinetics and thermodynamics of the
oxazolidine formation and cleavage may be modified advantageously.
Even though a co-solvent such as dimethylformamide (DMF) or
N-methylpyrrolidone (NMP) may be used to assist oxazolidine
formation, the resin is washed before elution so there is no
DMF/NMP in the eluate to interfere with a polishing purification
step involving reversed phase high pressure liquid chromatography
(HPLC). Tris(carboxyethyl)phosphine (TCEP) but not dithiothreitol
(DTT) is compatible with oxazolidine formation and helps prevent
disulfide bond formation between a bound full-length polypeptide
and a truncated impurity. Of course, peptides possessing N-terminal
Thr or Ser made by recombinant DNA techniques or found in nature,
can benefit from purification by covalent capture.
[0134] Polypeptides Not Possessing N-Terminal Cys, Thr or Ser: Use
of a Covalent Capture Tag
[0135] Polypeptides which do not possess Cys, Thr or Ser in the
N-terminal position may nonetheless react with certain aldehydes
under certain conditions (Tam et al. 1995 Int. J. Peptide Protein
Res. 45:209-216). However, the reactions are not very useful for
polypeptide purification by covalent capture since they are far
from quantitative, or they are irreversible, or they are too slow
or they require harsh conditions. To deal with such cases, which
form the majority of polypeptides and proteins, an auxiliary
chemical group may be attached to the N-terminus, or close to the
N-terminus, whose sole function is to act as a purification
<<handle>> (covalent capture tag). Of course, it is
usually desirable to be able to remove such a tag after it has
served its purpose. In order to avoid solubility problems
associated with fully-protected polypeptides, and in order to
exploit the full range of purification techniques, the tag must
remain attached to the polypeptide during the final post-synthetic
deprotection and cleavage step (from the synthesis resin). In
principle, such a tag need not function by covalent reaction
between the polypeptide and the purification matrix (generally a
gel, resin or other form of solid support), but it is better if it
does as this then avoids problems with very hydrophobic groups
(Tmob, biotin) or expensive tags such as peptide immunoaffinity
tags. While such a covalent approach has been shown to be a very
effective purification step (e.g. Funakoshi et al. 1991 Proc. Natl.
Acad. Sci. USA 88:6981-6985; Ball et al. 1995 J. Pept. Sci.
1:288-294; Roggero et al. 1997 FEBS Lett. 408:285-288), the
reagents and the conditions employed to remove the affinity tags
have been harsh: 5% ammonium hydroxide, 5% triethylamine or
cyanogen bromide in 70% trifluoroacetic acid, respectively, so
deamidation and other side reactions are a problem. In the case of
recombinant DNA-derived polypeptides, affinity tags such as
oligo-His, once they have served their purpose, are sometimes
removed by cleavage of the polypeptide chain with an endoprotease,
a process which proceeds under mild conditions but requires an
expensive reagent (the enzyme) and is sometimes difficult to drive
to completion.
[0136] Removal of the Covalent Capture Tag
[0137] While not always necessary, it is usually desirable to
remove the purification handle (capture tag) once it has served its
purpose.
[0138] Examples of cases where removal of the tag would not be
required include those where the tag was to be used in a subsequent
labelling step in solution, such a thiazolidine formation between
an N-terminal Cys polypeptide and an aldehyde-containing reporter
group (e.g. Zhang & Tam 1996 Analyt. Biochem. 233:87-93); or
amide bond formation between an N-terminal Cys polypeptide and a
thioester-containing reporter group (e.g. Kochendoerfer, G. G.
& Kent, S. B. 1999 Curr. Opin. Chem. Biol. 3:665-671 "Chemical
protein synthesis", and Cotton, G. J. & Muir, T. W. 1999
Chemistry & Biology 6:R247-R256 "Peptide ligation and its
application to protein engineering"); or oxime formation after
oxidation of N-terminal Ser or Thr (e.g. "Polypeptide and protein
derivatives and a process for their preparation", Offord, R. E. and
Rose, K., European Patent EP 0 243 929 B1, 27 Sep. 1995).
[0139] Removal of the capture tag is potentially problematic,
because a polypeptide chain devoid of protection groups is quite
fragile to the conditions used to remove an auxiliary group which
had been designed to withstand the powerful post-synthetic
deprotection and resin-cleavage conditions. Up to now, three types
of reaction have been proposed to remove purification handles:
[0140] cyanogen bromide. This reagent cleaves at a Met residue
placed between purification tag and the N-terminal residue of the
polypeptide of interest. It requires vigorous removal conditions
such as BRCN in 70% formic acid for many hours, followed by
reduction of internal Met residues temporarily protected as the
sulfoxide. Removal can lead to formylation of Trp and deamidation
of Asn and Gln. [0141] basic and/or nucleophilic conditions. These
conditions are used to cleave an Fmoc-type
(Fluorenyl-methyl-oxycarbonyl) or Msc-type
(Methyl-sulfonyl-ethyl-oxycarbonyl) group. They can lead to
deamidation, to racemization and to elimination reactions
(formation of dehydroalanine) followed by addition reactions.
[0142] enzymatic cleavage. This requires introduction of, for
example, a Factor Xa cleavage site. Unfortunately, the enzyme is
expensive and, being a macromolecular reagent cleaving an unnatural
macromolecular substrate, does not always cleave efficiently.
[0143] Removal of the Covalent Capture Tag by Periodate
Oxidation
[0144] It is known (Geohegan et al. 1979 "Reversible reductive
alkylation of amino groups in proteins", Biochemistry 18:5392-5399;
Feeney, R. E. 1987 "Chemical modification of proteins: comments and
perspectives", Int. J. Peptide Protein Res. 29:145-161) that
alpha-hydroxy-aldehydes (e.g. sugars) and alpha-hydroxy-ketones can
be attached to protein amino groups by reductive alkylation
(reaction 1) and then removed by periodate oxidation (reaction 2),
e.g.:
R--CH(OH)--CH.dbd.O+NH.sub.2--R'.fwdarw.R--CH(OH)--CH.sub.2--NH--R'
(1)
R--CH(OH)--CH.sub.2--NH--R'+HIO.sub.4.fwdarw.R--CHO+CH.sub.20O+NH.sub.2--
-R' (2)
[0145] Alternatively to (1), alkylation is a possibility:
R--H(OH)--CH.sub.2Br+NH.sub.2--R'.fwdarw.R--CH(OH)--CH.sub.2--NH--R'
(3)
R--CH(OH)--CH.sub.2NH.sub.2+BrCH.sub.2--CO--R'.fwdarw.R--CH(OH)--CH.sub.-
2--NH-etc (4)
[0146] In cases 1 and 3 di-derivatization of the amine should be
avoided, since [R--CH(OH)--CH.sub.2].sub.2N--R' is not cleaved by
periodate. Steric hindrance (e.g. through protection of the OH
group) during reductive alkylation or alkylation can help to avoid
such di-derivatization. A ketone may be used in place of the
aldehyde in 1, and a secondary bromide or epoxide in place of the
primary bromide in 3. For our application, R needs to contain a
group capable of binding to an affinity column, or we can use the
product R--CH(OH)--CH.sub.2--NH--R' itself to form an oxazolidine
with a capture resin which possesses carbonyl functions.
[0147] The periodate oxidation of a 1,2-amino-ol such as the
product of reaction 2 takes place under very mild conditions which
do not damage proteins. Indeed, this is the same procedure used to
oxidize an N-terminal Ser (R.dbd.H) or Thr (R.dbd.CH.sub.3) residue
of a protein to a glyoxylyl function:
NH.sub.2--CH[CH(R)OH]--CO--R'+HlO.sub.4+O.dbd.CH--CO--R'+NH.sub.3+R--CH.-
dbd.O (5)
a procedure which is known not to damage proteins (e.g.
"Polypeptide and protein derivatives and a process for their
preparation", Offord, R. E. and Rose, K., European Patent EP 0 243
929 B1, 27 Sep. 1995). Thus, a capture tag which is stable to the
post-synthetic cleavage/deprotection conditions, and has an
appropriate structure, may nevertheless be removed under very mild
conditions by periodate oxidation.
[0148] Structures of Capture Tags Removable by Periodate
Oxidation
[0149] In order for the periodate oxidation reaction to proceed
specifically and under mild conditions, a hydroxy group or an amino
group (not a thiol group) must be placed on a carbon vicinal to the
carbon which is directly attached to the alpha-amino group of the
first amino acid residue of the polypeptide. A thiol group is not
satisfactory in this position as treatment with periodate leads to
oxidation of the sulfur and subsequent failure to cleave the
carbon-carbon bond. Thus, a capture tag removable by periodate
oxidation under mild conditions and attached to the first amino
acid residue has the structure:
T-CR(OH)--CR'(R'')--NH--CHR.sup.1CO-- (a)
or T-CR(NH.sub.2)--CR'(R'')--NH--CHR.sub.1CO-- (b)
or T-CO--CR'(R'')--NH--CHR.sup.1CO-- (c)
where T is a capture tag group capable of making a strong
interaction (non-covalent, or preferably covalent) with a
purification matrix; R, R' and R'' are preferably hydrogen (to
minimize steric hindrance) but can be an alkyl, aralkyl or aryl
group or cyclic; and R' is the side chain of the first amino acid
residue.
[0150] Examples of capture functions for T include: [0151] Aminooxy
(e.g. aminooxyacetyl) group, which forms an oxime bond with a
carbonyl (aldehyde or ketone) resin. It is difficult to reverse
oxime formation, so when T incorporates the aminooxy function, it
is more convenient to release the peptide from the capture resin by
periodate cleavage of the linker. [0152] 1-amino-2-thiol (such as
Cys), which forms a thiazolidine with a carbonyl (aldehyde or
ketone) resin. [0153] 1-amino-2-ol (such as Thr), which forms an
oxazolidine with a carbonyl (aldehyde or ketone) resin. [0154]
Simply exploit the 1,2-amino-ol of
R--CH(OH)--CH.sub.2--NH--CHR.sup.1CO-- itself with an appropriate
carbonyl group on a resin to form an oxazolidine. [0155]
1,2-dithiol, which forms a dithioacetal with a carbonyl (aldehyde
or ketone) resin. [0156] 1-thio-2-ol, which forms an oxathioacetal
with a carbonyl (aldehyde or ketone) resin. [0157] 1,3-dithiol or
1-thio-3-ol, which form the corresponding 6-membered heterocycle
with a carbonyl (aldehyde or ketone) resin.
[0158] Boronate gels offer a potential alternative to
thiazolidine/oxazolidine chemistry for the capture of polypeptides
equipped with vicinal diols as covalent capture tag groups, such as
HO--CH.sub.2--CH(OH)--CH.sub.2--NH--CHR.sup.1--CO-etc. However,
they are expensive (but can be regenerated and reused), the
commercially available ones are not compatible with organic
solvents (but one could imagine a version based on
PEGA-NH--COCH.sub.2CH.sub.2CO--NH--C.sub.6H.sub.4-m-B(OH).sub.2),
and binding normally requires operation at pH 8 which would lead to
problems of mixed disulfides formed between captured peptide and
unwanted (capped) chains; avoiding mixed disulphides with excess
TCEP would be expensive.
[0159] Solid Phase Peptide Synthesis with Covalent Capture Tags
[0160] To avoid the difficulty of performing reductive alkylation
or alkylation reactions (1 and 3) "blindly" on the polypeptidyl
resin (we must avoid di-derivatization of the amine, since
(R--CH(OH)--CH.sub.2--).sub.2N--R' is not cleaved by periodate), it
is preferable to synthesize a set of protected amino acid
derivatives to be used to introduce the final residue during
automated solid phase synthesis. Such acylation reactions can be
made nearly quantitative. For example:
NH.sub.2--CH.sub.2--CH.sub.2--CH(OH)--CO.sub.2H (available from
Aldrich) may be amino protected with the Boc group, hydroxy
protected with the benzyl group (for Boc chemistry), coupled
through its carboxy group to HN(Me)OMe, reduced to the aldehyde
with LiAlH.sub.4, and reduced and alkylated to the amino group of a
side-chain protected amino acid. The secondary amine formed is
protected with the benzyloxycarbonyl group (known as the Z group),
resulting in formation of
Boc-NH--CH.sub.2--CH.sub.2--CH(OBzl)--CH.sub.2N(Z)-CH(R')CO--OH.
This protected amino acid derivative is coupled to the protected
polypeptidyl resin as the final residue. After capping any
unreacted amino groups and removal of the Boc group, a capture tag
group (e.g. Cys or Thr) is coupled prior to cleavage and
deprotection, which produces:
H-Cys/Thr-NH--CH.sub.2--CH.sub.2--CH(OH)--CH.sub.2NH--CH(R.sup.1)CO-poly-
peptide
[0161] Alternatively, a compound such as
Boc-Cys(Bu.sup.t)-NH--CH.sub.2--CH.sub.2--CH(OBu.sup.t)-CH.sub.2N(Boc)-CH-
(R.sup.1)C--OH may be used to introduce (i) a capture tag group
(Cys), (ii) a periodate-cleavable linker, and (iii) the N-terminal
amino acid residue, in one acylation step and to a polypeptide
which has been elongated by either Fmoc or Boc chemistry.
[0162] After purification on an appropriate aldehyde or ketone
support through covalent capture (thiazolidine or oxazolidine
formation), the released tagged polypeptide is treated with
periodate to liberate the target: H--NH--CH(R.sup.1)CO-polypeptide.
Thiol groups (but not disulfides) of any internal Cys residues
would react rapidly with periodate and must be temporarily blocked,
e.g. through the acetamidomethyl (Acm) group or other groups known
to be stable to liquid hydrogen fluoride, or through reversible
disulfide formation, or through oxidative refolding of the
polypeptide chain which forms intramolecular disulfide bonds.
Examples of suitable protecting groups for Cys are, besides the Acm
group, the S-Phacm group, the S-Smn group, and the S-Npys group
(Methods in Enzymology Vol. 289 p 205, Academic Press 1997, New
York). Tagged N-terminal Gly can be created on-resin from
BrCH.sub.2CO-peptide and unprotected
HO--CH.sub.2--CH.sub.2--NH.sub.2 (which is used anyway to remove
the formyl group from Trp).
[0163] HPLC Polishing Step
[0164] After release from the covalent capture resin, whether or
not a capture tag is present or has been removed, a final
purification (polishing) step is preferred, as a small amount of
full-length material can be damaged during deprotection (e.g.
alkylation of a Trp residue). Such polishing is conveniently
performed by reversed phase high pressure liquid chromatography
(HPLC). Nonetheless, it is much easier to purify by HPLC and to
lyophilize the relatively small amounts of released full-length
material than to try to deal directly by HPLC with large amounts of
crude material. Covalent capture tagging is thus much better than
simple chromatographic (diagonal HPLC tags) as it avoids HPLC and
lyophilization of bulk crude polypeptide. As noted above, the
amount of full-length product after a long synthesis is sometimes
only a small proportion of the total, e.g. 37% after 100 cycles at
99%, and only 0.59% after 100 cycles at 95%.
[0165] N-Terminal Prolyl, Acetyl or Pyroglutamyl Polypeptides
[0166] Traceless, periodate-removable capture tags can be put on a
Lys side-chain using building blocks such as
Boc-Lys[N(2ClZ)CH.sub.2--CH.sub.2--O-Bzl]-OH. If the modified Lys
is close to the N-terminus and subsequent couplings are
quantitative, this permits purification by covalent capture of
N-blocked peptides (e.g. N-terminal acetyl or pyroglutamylpeptides)
and of N-terminal Pro peptides (which, if tagged directly, would
fail to be deprotected by periodate).
[0167] Thioesters
[0168] Although Boc chemistry is generally employed to prepare
polypeptide thioesters, several groups have shown that it is also
possible to use Fmoc chemistry: Li et al. (1998, Tetrahedron Lett.
39:8669-8672), Ingenito et al. (1999, J. Am. Chem. Soc.
121:11369-11374), Youngsook et al. (1999, J. Am. Chem. Soc.
121:11684-11689, and Alsina et al. (1999 J. Org. Chem.
64:8761-8769). The Boc thioester methodology has been simplified by
Hackeng et al. (1999) Proc. Natl. Acad. Sci. USA 96:10068-10073,
and used to prepare fully active human secretory phospholipase A(2)
from 4 segments. The peptide ligation reaction itself may be
performed on the solid phase, and up to eight polypeptides have
been linked together in this way into a single protein chain (cited
in Kochendoerfer & Kent). In the case of polypeptide-thioesters
to be used for ligation, N-terminal Cys cannot be used as a
purification tag or the peptide thioester would cyclize. As an
alternative to thioacids (see above), a Thr capture tag group may
be used. After purification of Thr-linker-peptidel-thioester on an
aldehyde column and ligation with Cys-peptide2 to produce:
H-Thr-NH--CH.sub.2--CH.sub.2--CH(OH)--CH.sub.2NH--CH(R)CO-Cys-peptide1-C-
ys-peptide2
[0169] Any free thiol side chains are protected with
S-nitropyridylsulfenyl prior to cleavage of the linker with
periodate. Treatment with DTT then leaves
H-Cys-peptide1-Cys-peptide2. Schemes for ligation of peptides on
the solid phase have been described (Canne et al. 1999 J. Am. Chem.
Soc. 121:8720-8727), but involve rather harsh release conditions
(pH 14). Use of a periodate-cleavable traceless capture tag would
be helpful here also, and can be made based on the chemistry
described above. For example, the
CH.sub.3COCH.sub.2CH.sub.2CO--NHCH.sub.2CH.sub.2SO.sub.2CH.sub.2CH.sub.2O-
CO-- capture tag of Canne et al. may be replaced with
CH.sub.3COCH.sub.2CH.sub.2CO--NH--CH.sub.2--CH.sub.2--CH(OH)--CH.sub.2NH--
-CHR.sup.1--CO--, where NH--CHR.sup.1--CO is the first residue of
the peptide.
EXAMPLES
Example 1
[0170] The peptide YAKYAKL was prepared by standard techniques on
an ABI 430A synthesizer using Boc chemistry with in situ
neutralization and HBTU activation (Methods in Enzymology Vol.
289). A portion of the resin was extended with Boc-Cys(4MeBzl), and
both portions were then cleaved and deprotected (HF with 5%
p-cresol, 0.degree. C., 1 h). After precipitation with cold
diethylether, the peptides were purified by preparative HPLC and
characterized by electrospray ionization mass spectrometry. An
aliphatic aldehyde column was prepared starting with Amino-PEGA
resin (NovaBiochem, Switzerland), acylating it with succinic
anhydride and activating it with carbonyl-dimimidazole as
previously described (Rose, K. and Vizzavona, J. 1999, J. Am. Chem.
Soc. 121:7034-7038), then aminolyzing with aminoacetaldehyde
diethylacetal (Fluka, Buchs, Switzerland; 4 ml diluted with 4 ml
DMF and made 0.5 M in hydroxybenztriazole). After thorough washing
with DMF, the resin was deacetalized by treatment with
water/trifluoroacetic acid (1:1, v/v) for 2 hours at room
temperature. The resulting aldehyde resin was washed thoroughly
with DMF, then with water/acetonitrile (1:1, v/v), then with an
acetate buffer (0.2 M, sodium counter-ion, 50% in acetonitrile, 2
mM in EDTA, pH 4.5). To one volume of resin, 0.9 volume of peptide
solution (1 mg/ml each of YAKYAKL and CYAKYAKL in the acetate
buffer) was added with mixing. After 1 h at room temperature
(22.degree. C.), an aliquot of the supernatant was analyzed by HPLC
to assess the extent of covalent capture of the Cys peptide. After
washing away the unbound Tyr peptide with the acetate buffer
solution, bound peptide was released from the aldehyde resin by
reversal of thiazolidine formation with 1% trifluoroacetic acid
(TFA) in 50% acetonitrile, 0.1 M aminooxyacetic acid. After 24 h at
room temperature, essentially all of the bound Cys peptide had been
released into the supernatant, and its presence was confirmed by
analysis by HPLC and mass spectrometry. FIG. 1 shows the data
obtained. Similar experiments with 5 mg/ml peptide instead of 1
mg/ml were also successful.
Example 2
[0171] A similar experiment to Example 1 was performed except that
an aromatic aldehyde resin (Amino-PEGA resin which had been
acylated with formyl-benzoyl hydroxysuccinimide ester) was used in
place of the aliphatic aldehyde resin. Similar results were
obtained, although capture of the CYAKYAKL was slower (18 h at room
temperature).
Example 3
[0172] A similar experiment to Example 1 was performed except that
an N-terminal Thr peptide (TYAKYAKL, 5 mg/ml) replaced the
N-terminal Cys peptide, and a phosphate buffer (0.1 M, 50% in
acetonitrile, pH 7.0) replaced the acetate buffer. Capture reached
equilibrium (about 50% of the peptide was bound to the resin) after
24 h. Elution was achieved by incubation with 0.1 M dithiothreitol
in 50% acetonitrile, 1% trifluoroacetic acid, 20 hours.
Example 4
[0173] A similar experiment to Example 3 was performed except that
an N-terminal Ser peptide (SYAKYAKL) was used in place of the
N-terminal Thr peptide. Capture reached equilibrium (about 10%)
after 24 hours under the particular conditions used.
Example 5
[0174] A similar experiment to Example 2 was performed except that
the N-terminal Cys peptide had the sequence
CAVVFVTRKNRQVSANPEKKAVREYINSLELA and the control sequence was
ACAVVFVTRKNRQVSANPEKKAVREYINSLELA. Capture, purification from the
control sequence and elution were successful.
Example 6
[0175] Protection of Primary Amine and Hydroxyl Function with
Boc.sub.2O and BrBzl Respectively
[0176] To 5 g (42 mmol) (S)-(-)-4-amino-2-hydroxybutryric acid
(product number 46735-9, Aldrich Chemical Co.) was added 50 ml
dioxane and 150 ml water. The pH was raised to 11 with 105 ml 1 M
NaOH, whereupon 13.7 g Boc.sub.2O (63 mmol) was added and the
suspension mixed briskly at room temperature overnight. The next
day the solution, which had become clear, was acidified with 1 M
HCl to pH 2 and the dioxane removed by rotary evaporation. The
solution was extracted 10 times with 50 ml portions of
dichloromethane, the pooled organic phases were dried over
anhydrous sodium sulfate, filtered and evaporated to afford an oil,
which was dried in a dessicator overnight. Yield 8.7 g (94%). To
this oil (39 mmol) was added 50 ml dry tetrahydrofuran (THF) and
the flask cooled with ice bath to about 0.degree. C. Temperature
control is important to avoid formation of the benzyl ester as well
as the desired benzyl ether. Sodium hydride (2.1 g, 85.8 mmol) was
added in portions and allowed to react for 15 min. Then 5.1 ml (43
mmol) benzyl bromide was added, still at 0.degree. C. After
stirring overnight, the temperature was allowed to rise to room
temperature and the THF was removed by rotary evaporation. Water
was added (150 ml) and the aqueous phase was washed twice with
ether (2.times.50 ml) before being acidified with 100 ml 1 M
KHSO.sub.4. The solution was extracted 5 times with 50 ml portions
of ethyl acetate, the organic phase was dried over sodium sulfate,
filtered and rotary evaporated to afford 9.6 g crude product.
Boc-NH--CH.sub.2--CH.sub.2--CH(O-benzyl)-CO.sub.2H. Yield: 80%. MS
m/z 309.50 found (m/z 309.36 calculated).
[0177] A portion of this protected acid (7.4 g, 24 mmol) was mixed
with N-methyl-methoxamine hydrochloride (3.5 g, 36 mmol), then the
coupling agent BOP (10.1 g, 23 mmol, in 40 ml N,N-dimethylfornamide
(DMF)) was added. N,N-diisopropyl-ethylamine (14.4 ml, 84 mmol) was
added, which led to warming and dissolution of the reactants. After
incubation overnight at room temperature, the solution was diluted
with 150 ml ethyl acetate and the organic phase washed with
saturated sodium bicarbonate solution (3 times, 150 ml each time),
once with 150 ml brine, 3 times with 1 M KHSO.sub.4 and once more
with 150 ml brine. After drying over sodium sulfate and filtering,
the organic phase was rotary evaporated to afford 8.3 g (100%)
product as an oil. Analysis by electrospray ionization mass
spectrometry gave signals at 374. 8 (M+sodium), 352.8 (M+proton),
252.5 (base peak, M+proton minus Boc+H), as expected for
Boc-NH--CH.sub.2--CH.sub.2--CH(O-benzyl)-CO--N(CH.sub.3)--OCH.sub.3.
[0178] Reduction to the Aldehyde
[0179] Reduction to the aldehyde was achieved by dissolving this
hydroxamate (2 g, 5.7 mmol) in 50 ml dry THF, cooling in ice to
about 0.degree. C., then adding LiAIH4 in portions (about 300 mg, 8
mmol) over a period of about an hour to the stirred solution, still
at 0.degree. C. Reaction progress was followed by thin layer
chromatography (silica gel 60 F.sub.254, ethyl acetate/hexane 1:1,
revelation with charring reagent). When TLC showed quantitative
conversion to the more hydrophobic material, 150 ml ethyl acetate
was added and stirring was continued for one hour. Then 150 ml
brine was added. Ten minutes later, the organic phase was
separated, washed twice with 150 ml portions of brine containing 50
ml of 1N potassium hydrogen sulfate to facilitate removal of
aluminium salts, dried over sodium sulfate, filtered, and
concentrated in vacuo to give a clear oil. Yield: 100%.
[0180] Reductive Alkylation
[0181] The corresponding aldehyde (706 mg, 2 mmol) was dissolved in
a mixture of methanol-acetic acid (99:1, 25 ml) containing the
commercial compound HCl.H-Gly-OMe (500 mg, 4 mmol). Sodium
cyanoborohydride (378 mg, 6 mmol) was dissolved in 10 ml of
methanol-acetic in order to add it dropwise during 10 min. After 3
days, a further amount of cyanoborohydride reducing agent (100 mg,
1.6 mmol) was added in the solution. After one week under stirring,
the reaction was complete according to HPLC. A saturated solution
of sodium bicarbonate (20 ml) was added under vigorous vortex, then
the methanol was removed in vacuo. Ethyl acetate (150 ml) and
saturated bicarbonate solution (150 ml) were added and the mixture
shaken. The separated organic layer was washed successively with
saturated bicarbonate solution (2 portions of 100 ml), then brine
(1.times.100 ml), dried over sodium sulfate and then concentrated
in vacuo. The product
Boc-NH-CH.sub.2--CH.sub.2--CH(O-benzyl)-CH.sub.2NHCH.sub.2CO--OMe
was purified by HPLC. Yield: 300 mg, 41%. MS m/z 366.14 found (m/z
366.45 calculated).
[0182] Protection of the Secondary Amine
[0183] To a solution of the previous compound (300 mg, 0.8 mmol)
dissolved in THF (8 ml) was added
N-(benzyloxycarbonyloxy)succinimide (597 mg, 2.4 mmol),
diisopropylethylamine (550 .mu.l, 3.2 mmol). After stirring
overnight, the reaction was concentrated in vacuo before mixing in
saturated bicarbonate solution (60 ml) and ethyl acetate (60 ml)
during 5 min. The organic layer was washed with saturated
bicarbonate solution (2 portions each of 60 ml), NaCl saturated
water (1.times.60 ml), 1N potassium hydrogen sulfate (2.times.60
ml), brine (1.times.60 ml), was dried over sodium sulfate and then
concentrated in vacuo. The residue, triturated with isopropanol
then dried in vacuo, gave a white powder. Yield: 400 mg, 100%. MS
m/z 500.61 found (m/z 500.59 calculated).
[0184] Saponification
[0185] The fully protected compound (400 mg, 0.8 mmol) was
saponified with aqueous NaOH 2N solution (4.8 ml, 9.6 mmol) in THF
(6 ml) at cold water temperature. The hydroxide sodium solution was
added dropwise. The reaction was checked by HPLC. After stirring
overnight, the solution was acidified with 1N potassium hydrogen
sulfate to pH 1 and THF removed by rotary evaporation. Then,
ethylacetate (100 ml) was added to form an emulsion under stirring
and the organic phase washed with 1N potassium hydrogen sulfate
(2.times.100 ml), brine (100 ml), dried over sodium sulfate and
then concentrated in vacuo. The residue gave a yellow oil. Yield:
300 mg, 77%. MS m/z 486.77 found (m/z 486.56 calculated).
Example 7
[0186] The protected polypeptide sequence
GCAVVFVTRKNRQVSANPEKKAVREYINSLELA was synthetized by standard Boc
SPPS on a PAM resin. The last Glycine residue was introduced as
Boc-NH--CH.sub.2--CH.sub.2CH(OBzl)CH.sub.2N(Z)CH.sub.2COO activated
with DCC/HOAt for 30 minutes before coupling with the resin. After
capping with acetic anhydride and Boc removal with TFA,
Boc-Cys(pMeBzl)-OH was introduced and the resulting
Boc-Cys(pMeBzl)-NH--CH.sub.2--CH.sub.2CH(OBzl)CH.sub.2G(N.sup..alpha.Z)GC-
AVVFVTRKNRQVSANPEKKAVREYINSLELA-OH was cleaved with HF/cresol. The
crude material
H-Cys-NH--CH.sub.2--CH.sub.2CH(OH)CH.sub.2GGCAVVFVTRKNRQVSANPEKK-
AVREYINSLELA-OH (0.25 .mu.moles in 100 .mu.l) solubilized in
acetate buffer (0.2 M, sodium counter-ion, 50% in acetonitrile, 2
mM in EDTA, pH 4.5) was added to an equal volume of the aliphatic
aldehyde resin of Example 1 equilibrated in the same buffer.
Aliquots of the supernatant were analysed by HPLC after 1, 5 and 16
hours. The capture of the correct sequence was completed in 16
hours, while the impurities present failed to interact with the
resin. The resin was washed with the acetate buffer (4 portions
each of 10 bed volumes). No significant amount of the wanted
correct peptide leaked in the washes, which were analysed by HPLC.
Bound peptide was released from the aldehyde resin by reversal of
thiazolidine formation with 1% trifluoroacetic acid (TFA) in 50%
acetonitrile, 0.1 M aminooxyacetic acid. After 24 h at room
temperature, essentially all of the bound Cys peptide had been
released into the supernatant, and its presence and purity was
confirmed by HPLC and mass spectrometry analyses.
Example 8a
Cleavage of Tagged Peptide
[0187]
tCys-NH--CH.sub.2--CH.sub.2CH(OH)CH.sub.2GGCAVVFVTRKNRQVSANPEKKAVRE-
YINSLELA after elution from the aldehyde resin (Example 7) was
desalted by HPLC (expected mass 3938.6, experimental 3942.4). The
peptide was treated with 2 equivalents of TCEP (acetate buffer, 0.2
M, pH 4.5) to completely reduce the cysteines, and treated with 20
equivalents of 2,2'dithiodipyridine for 2 hours. The resulting
Cys(Spy)-NH--CH.sub.2--CH.sub.2CH(OH)CH.sub.2GGC(Spy)AVVFVTRKNRQVSANPEKKA-
VREYINSLELA was purified by RP-HPLC (Expected mass 4156,
experimental 4158). The purified material was treated with 10
equivalents of NaIO.sub.4 in imidazole hydrochloride buffer pH 7 in
the presence of 50 equivalents of Methionine. After 10 minutes the
reaction was stopped with excess ethylene glycol, acidified with
acetic acid and immediately purified by RP-HPLC.
H.sub.2N-GGC(Spy)AVVFVTRKNRQVSANPEKKAVREYINSLELA-OH was reduced
with TCEP and the expected material was recovered after a desalting
step (Expected mass 3748, experimental 3945).
Examples 8b
Cleavage of Tagged Dipeptides
[0188] A series of
NH.sub.2--CH.sub.2--CH.sub.2CH(OH)CH.sub.2--NH--X.sub.1--X.sub.2
tag-dipeptides (X.sub.1, X.sub.2 represent two amino acid residues)
were synthesised in solution phase by direct reductive alkylation
to evaluate the effect of different amino acids on the periodate
oxidation rate. Boc-NH--CH.sub.2--CH.sub.2CH(OBzl)CHO (1 eq.) and
(in separate experiments) H.sub.2N-Asp-Phe-NH.sub.2;
H.sub.2N-Met-Phe-OH; H.sub.2N-Leu-Phe-OH and H.sub.2N-Ile-Phe-OH (2
eq.) were treated with 3 eq. of NaBH.sub.3CN. Each
NH--CH.sub.2--CH.sub.2CH(OH)--CH.sub.2--NH--X.sub.1--X.sub.2 was
purified by RP-HPLC after Boc and Bzl removal with
trifluoromethanesulfonic acid in TFA under standard conditions.
Each tag-dipeptide was treated at room temperature (22.degree. C.)
with different equivalents of NaIO.sub.4 in the presence of 50
equivalents of Methionine in 50 mM Imidazole pH 6.95. The reactions
were stopped after 5 minutes. The extent of tag removal was
evaluated by RP-HPLC. In this series of experiments, X.sub.2 was
always Phe.
TABLE-US-00001 Equivalents of NaIO.sub.4 necessary X.sub.1 Amino
acid to completely remove the tag Methionine 20 Leucine 30 Aspartic
acid 5 Isoleucine 30 Glycine 5
Examples 9
Synthesis of Acetaldehyde-Sephadex CM C50 Resin
[0189] Sephadex CM C50 (Pharmacia) (100 mg) with a substitution of
4.5 meq/g dry resin was swollen and degassed in H.sub.2O under
vacuum for 20 minutes giving a final bed volume of 2 ml. The resin
was treated with 10 ml of 200 mM sodium phosphate buffer, pH 6.5,
and then equilibrated with H.sub.2O. The carboxylic functions were
activated as N-Hydroxysuccinimide esters by treating the resin for
8 minutes with a water solution of N-Hydroxysuccinamide (0.9
mmoles) and 1'-Ethyl-(3'-dimethylaminopropyl)-carbodiimide.HCl (1.8
mmoles). The activation solution was eliminated and the resin
rapidly rinsed with H.sub.2O. A solution of amino acetaldehyde
diethyl acetal (4.5 mmoles) in 5 ml of a 200 mM
2-Morpholino-ethanesulfonic acid monohydrate buffer, was prepared
and brought to pH 6.4 with 3 N HCl. The resin was incubated under
gentle mixing with this solution for 1 hour. After 1 hour the resin
was washed with a solution of ammonium acetate 1 M, pH 7 for 10
minutes. Acetal protection of the aldehyde function was achieved by
treating the resin with a solution of 10 mM HCl for 10 minutes. The
resin was equilibrated with a solution 200 mM sodium acetate, 2 mM
EDTA, pH 4.5.
Example 10
Covalent Capture of CYAKYAKL with Acetaldehyde-Sephadex CM C50
Resin
[0190] 1 mg of the CYAKYAKL peptide was solubilized in 100 .mu.l of
a buffer composed of 200 mM sodium acetate, 2 mM EDTA, 6 M
guanidine hydrochloride, 2 mM DIT, pH 4.5 (Binding buffer). The
solution was incubated with 20 .mu.l of the
Sephadex-aminoacetaldehyde resin for 48 hours. The supernatant was
removed and the resin was washed 3 times with 1 ml of the binding
buffer. The bound peptide was eluted with 200 .mu.l of a solution
200 mM of O-methythydroxylamine hydrochloride, pH 3.5 for 16 hours.
The extent of capture was estimated by RP-HPLC analysis of the
solution before capture, that after 48 hours, the washing solution,
and the eluate. After 48 hours of incubation, 5% of the peptide was
still present in the supernatant. The washing solution did not
contain any peptide. The elution solution contained 0.9 mg of
CYAKYAKL. The efficiency of the recovery was thus estimated to be
about 90%.
Example 11
Covalent Capture of Rantes10-68 with Acetaldehyde-Sephadex CM C50
Resin
[0191] Rantes 10-68, a truncated version of human Rantes possessing
N-terminal cysteine, was expressed in Escherichia coli by
recombinant techniques. After cell lysis, the inclusion bodies
fraction was obtained by high speed centrifugation. The precipitate
corresponding to 1 liter of bacterial culture was solubilized in 5
ml of binding buffer, composed of 200 mM sodium acetate, 2 mM EDTA,
6 M guanidine hydrochloride, 2 mM DTT, pH 4.5. The solubilized
product was incubated with 1 ml of the Sephadex-acetaldehyde resin
at 4.degree. C. for 48 hours. The supernatant was removed and the
resin was washed 3 times with 5 ml of the binding buffer to remove
unbound material. The bound protein was eluted with 3 ml of a
solution of 200 mM of O-methylhydroxylamine hydrochloride, 1 mM
TCEP, with 20 .mu.l of glacial acetic acid at pH 3.5 for 16 hours.
The resin after removal of the eluted material was further washed
with 2 ml of the same elution buffer for 10 minutes and the two
fractions were combined. The eluted material was analyzed by
RP-HPLC. The major product eluted, representing 90% of the
integrated area of the chromatogram, was analyzed by MALDI-TOF mass
spectrometry, and corresponded to the expected material (expected
mass 6915.14 Da, experimental 6916.68 Da). The solution was
dialyzed against 1% acetic acid, then lyophilized. The total amount
recovered from 1 liter of bacterial culture was 1.5 mg.
* * * * *