U.S. patent application number 17/558525 was filed with the patent office on 2022-09-01 for solid phase peptide synthesis processes and associated systems.
This patent application is currently assigned to Massachusetts Institute of Technology. The applicant listed for this patent is Massachusetts Institute of Technology. Invention is credited to Andrea Adamo, Patrick Louis Heider, Klavs F. Jensen, Bradley L. Pentelute, Mark David Simon.
Application Number | 20220275021 17/558525 |
Document ID | / |
Family ID | 1000006333191 |
Filed Date | 2022-09-01 |
United States Patent
Application |
20220275021 |
Kind Code |
A1 |
Simon; Mark David ; et
al. |
September 1, 2022 |
SOLID PHASE PEPTIDE SYNTHESIS PROCESSES AND ASSOCIATED SYSTEMS
Abstract
Systems and processes for performing solid phase peptide
synthesis are generally described. Solid phase peptide synthesis is
a known process in which amino acid residues are added to peptides
that have been immobilized on a solid support. In certain
embodiments, the inventive systems and methods can be used to
perform solid phase peptide synthesis quickly while maintaining
high yields. Certain embodiments relate to processes and systems
that may be used to heat, transport, and/or mix reagents in ways
that reduce the amount of time required to perform solid phase
peptide synthesis.
Inventors: |
Simon; Mark David; (Boston,
MA) ; Pentelute; Bradley L.; (Cambridge, MA) ;
Adamo; Andrea; (Cambridge, MA) ; Heider; Patrick
Louis; (Midland, MI) ; Jensen; Klavs F.;
(Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Massachusetts Institute of Technology |
Cambridge |
MA |
US |
|
|
Assignee: |
Massachusetts Institute of
Technology
Cambridge
MA
|
Family ID: |
1000006333191 |
Appl. No.: |
17/558525 |
Filed: |
December 21, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17127961 |
Dec 18, 2020 |
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17558525 |
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15602056 |
May 22, 2017 |
10889613 |
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17127961 |
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14776870 |
Sep 15, 2015 |
9695214 |
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PCT/US2014/017970 |
Feb 24, 2014 |
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15602056 |
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13833745 |
Mar 15, 2013 |
9169287 |
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14776870 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01J 8/00 20130101; B01J
19/00 20130101; C07K 1/045 20130101; C07K 1/061 20130101; C07K
1/042 20130101; C07K 1/08 20130101; C07K 1/04 20130101; B01J
19/0053 20130101 |
International
Class: |
C07K 1/04 20060101
C07K001/04; C07K 1/06 20060101 C07K001/06; C07K 1/08 20060101
C07K001/08; B01J 19/00 20060101 B01J019/00; B01J 8/00 20060101
B01J008/00 |
Claims
1-77. (canceled)
78. A process for adding amino acid residues to peptides,
comprising: merging a first stream comprising first amino acids
that comprise first protecting groups with a second stream
comprising a first amino acid activating agent such that the first
and second streams form a first mixed fluid comprising first
activated amino acids; exposing the first mixed fluid to a
plurality of peptides immobilized on a solid support such that
first amino acid residues are added to the plurality of peptides;
flowing a deprotection reagent to remove at least a portion of the
first protecting groups; merging a third stream comprising second
amino acids that comprise second protecting groups and a fourth
stream comprising a second amino acid activating agent to form a
second mixed fluid comprising second activated amino acids; and
exposing the second mixed fluid to the plurality of peptides
immobilized on the solid support such that second amino acid
residues are added to the plurality of peptides; wherein there is
no washing step between the first merging step and the second
merging step.
79. The process of claim 78, further comprising washing the solid
support with a washing solvent after the addition of the second
amino acid residues to the plurality of peptides.
80. The process of claim 78, further comprising flowing a second
deprotection reagent to remove at least a portion the second
protecting groups.
81. The process of claim 78, wherein the first protecting groups
and/or the second protecting groups comprise
fluorenylmethyloxycarbonyl protecting groups.
82. The process of claim 78, wherein first amino acid residues are
added to at least about 99% of the immobilized peptides after
exposing the first mixed fluid to the immobilized peptides.
83. The process of claim 78, wherein first amino acid residues are
added to at least about 99.9% of the immobilized peptides after
exposing the first mixed fluid to the immobilized peptides.
84. The process of claim 78, wherein second amino acid residues are
added to at least about 99.9% of the immobilized peptides after
exposing the second mixed fluid to the immobilized peptides.
85. The process of claim 78, wherein second amino acid residues are
added to at least about 99% of the immobilized peptides after
exposing the second mixed fluid to the immobilized peptides.
86. The process of claim 78, wherein the solid support is contained
within a packed column and/or a fluidized bed.
87. The process of claim 78, wherein the solid support comprises
polystyrene and/or polyethylene glycol.
88. The process of claim 78, wherein the solid support comprises a
resin.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 17/127,961, filed Dec. 18, 2020, and entitled
"Solid Phase Peptide Synthesis Processes and Associated Systems,"
which is a division of U.S. patent application Ser. No. 15/602,056,
filed May 22, 2017, and entitled "Solid Phase Peptide Synthesis
Processes and Associated Systems," which is a continuation of U.S.
patent application Ser. No. 14/776,870, filed Sep. 15, 2015, and
entitled "Solid Phase Peptide Synthesis Processes and Associated
Systems," which is a national stage filing under 35 U.S.C. .sctn.
371 of International Application Number PCT/US2014/017970, filed
Feb. 24, 2014, entitled "Solid Phase Peptide Synthesis Processes
And Associated Systems," which is a continuation-in-part of U.S.
patent application Ser. No. 13/833,745, filed Mar. 15, 2013, and
entitled "Solid Phase Peptide Synthesis Processes and Associated
Systems," each of which is incorporated herein by reference in its
entirety for all purposes.
TECHNICAL FIELD
[0002] Systems and processes for performing solid phase peptide
synthesis are generally described.
BACKGROUND
[0003] Solid phase peptide synthesis is a process used to
chemically synthesize peptides on solid supports. In solid phase
peptide synthesis, an amino acid or peptide is bound, usually via
the C-terminus, to a solid support. New amino acids are added to
the bound amino acid or peptide via coupling reactions. Due to the
possibility of unintended reactions, protection groups are
typically used. To date, solid phase peptide synthesis has become
standard practice for chemical peptide synthesis. The broad utility
of solid phase peptide synthesis has been demonstrated by the
commercial success of automated solid phase peptide synthesizers.
Though solid phase peptide synthesis has been used for over 30
years, fast synthesis techniques have not yet been developed.
Accordingly, improved processes and systems are needed.
SUMMARY
[0004] Solid phase peptide synthesis processes and associated
systems are generally described. Certain embodiments relate to
systems and methods which can be used to perform solid phase
peptide synthesis quickly while maintaining high yield. In some
embodiments, reagents can be heated, transported, and/or mixed in
ways that reduce the amount of time required to perform solid phase
peptide synthesis. The subject matter of the present invention
involves, in some cases, interrelated products, alternative
solutions to a particular problem, and/or a plurality of different
uses of one or more systems and/or articles.
[0005] In some embodiments, a process for adding amino acid
residues to peptides is provided. The process, in certain
embodiments, comprises heating a stream comprising amino acids such
that the temperature of the amino acids is increased by at least
about 1.degree. C.; and exposing the heated amino acids to a
plurality of peptides immobilized on a solid support, wherein the
heating step is performed prior to and within about 30 seconds of
exposing the heated amino acids to the peptides.
[0006] In certain embodiments, the process comprises providing a
plurality of peptides comprising protection groups, each peptide
immobilized on a solid support; performing a first amino acid
addition cycle comprising exposing amino acids to the immobilized
peptides such that an amino acid residue is added to at least about
99% of the immobilized peptides; and performing a second amino acid
addition cycle comprising exposing amino acids to the immobilized
peptides such that an amino acid residue is added to at least about
99% of the immobilized peptides. In some embodiments, the total
amount of time between the ends of the first and second amino acid
addition cycles is about 10 minutes or less and the protection
groups comprise fluorenylmethyloxycarbonyl protection groups and/or
the total amount of time between the ends of the first and second
amino acid addition cycles is about 5 minutes or less.
[0007] In certain embodiments, the process comprises providing a
plurality of peptides immobilized on a solid support; and exposing
activated amino acids to the immobilized peptides such that at
least a portion of the activated amino acids are bonded to the
immobilized peptides to form newly-bonded amino acid residues;
wherein an amino acid residue is added to at least about 99% of the
immobilized peptides within about 1 minute or less.
[0008] In certain embodiments, the process comprises flowing a
first stream comprising amino acids; flowing a second stream
comprising an amino acid activating agent; merging the first and
second streams to form a mixed fluid comprising activated amino
acids; and within about 30 seconds after merging the first and
second streams to form the mixed fluid, exposing the mixed fluid to
a plurality of peptides immobilized on a solid support.
[0009] In certain embodiments, the process comprises providing a
plurality of peptides comprising protection groups, each peptide
immobilized on a solid support; exposing a deprotection reagent to
the immobilized peptides to remove protection groups from at least
a portion of the immobilized peptides; removing at least a portion
of the deprotection reagent; exposing activated amino acids to the
immobilized peptides such that at least a portion of the activated
amino acids are bonded to the immobilized peptides to form
newly-bonded amino acid residues; and removing at least a portion
of activated amino acids that do not bond to the immobilized
peptides. In some embodiments, an amino acid residue is added to at
least about 99% of the immobilized peptides during the amino acids
exposing step. In certain embodiments, the total amount of time
taken to perform the combination of all of the deprotection reagent
exposing step, the deprotection reagent removal step, the activated
amino acid exposing step, and the activated amino acid removal step
is about 10 minutes or less and the protection groups comprise
fluorenylmethyloxycarbonyl protection groups and/or the total
amount of time taken to perform the combination of all of the
deprotection reagent exposing step, the deprotection reagent
removal step, the activated amino acid exposing step, and the
activated amino acid removal step is about 5 minutes or less.
[0010] Other advantages and novel features of the present invention
will become apparent from the following detailed description of
various non-limiting embodiments of the invention when considered
in conjunction with the accompanying figures. In cases where the
present specification and a document incorporated by reference
include conflicting and/or inconsistent disclosure, the present
specification shall control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] Non-limiting embodiments of the present invention will be
described by way of example with reference to the accompanying
figures, which are schematic and are not intended to be drawn to
scale. In the figures, each identical or nearly identical component
illustrated is typically represented by a single numeral. For
purposes of clarity, not every component is labeled in every
figure, nor is every component of each embodiment of the invention
shown where illustration is not necessary to allow those of
ordinary skill in the art to understand the invention. In the
figures:
[0012] FIG. 1 is a schematic illustration of a system for
performing peptide synthesis, according to one set of
embodiments;
[0013] FIGS. 2A-2D are, according to certain embodiments, (A) an
exemplary schematic diagram of a peptide synthesis system, (B) a
photograph of an exemplary peptide synthesis system, (C) a
chromatogram for a synthesized peptide Fmoc-ALFALFA-CONH.sub.2,
(SEQ ID NO: 1) and (D) an exemplary schematic diagram of a
reactor;
[0014] FIGS. 3A-3C are, according to one set of embodiments, (A)
chromatograms of LYRAG-CONH.sub.2 (SEQ ID NO: 2) peptides
synthesized with different activated amino acid exposing times, (B)
chromatograms of Fmoc-ALF-CONH.sub.2 peptides synthesized with
different activated amino acid exposing times, and (C) an exemplary
synthetic timeline;
[0015] FIGS. 4A-4D are, according to certain embodiments,
chromatograms and mass spectra for ACP (65-74) peptides (SEQ ID NO:
3) (A) synthesized using HATU at 60.degree. C. and (B) synthesized
using HBTU at 60.degree. C., (C) synthesized using HBTU at RT and
(D) synthesized using HBTU under batch conditions using the same
synthetic timeline;
[0016] FIGS. 5A-5B are, according to one set of embodiments, (A) a
chromatogram and mass spectrum for a synthesized PnIA (A10L)
peptide (SEQ ID NO: 4) and (B) a chromatogram and mass spectrum for
a synthesized HIV-1 PR (81-99) peptide (SEQ ID NO: 5);
[0017] FIGS. 6A-6E are, according to certain embodiments, total ion
current chromatographs for GCF peptides synthesized under various
conditions (A) 5, (B) 7, (C) 8, and (D) 4, as shown Table 1, and
(E) is an exemplary total ion current chromatograph for an
authentic Gly-D-Cys-L-Phe sample;
[0018] FIGS. 7A-7E are, according to certain embodiments, (A) an
exemplary scheme for the chemical ligation of an affibody protein
from three peptide fragments, (B) chromatogram and mass spectrum
for a first affibody fragment (SEQ ID NO: 6), (C) chromatogram and
mass spectrum for a second affibody fragment (SEQ ID NO: 7), (D)
chromatogram and mass spectrum for a third affibody peptide
fragment (SEQ ID NO: 8), and (E) chromatogram and mass spectrum for
the purified affibody;
[0019] FIGS. 8A-8F are, according to certain embodiments, (A-B)
total ion chromatograms for the N-terminal affibody fragment using
Fmoc N-terminal protecting groups and a traditional manual
arrangement using Boc N-terminal protecting groups, respectively
(sequences in FIGS. 8A and 8B correspond to SEQ ID NOs.: 9 and 10,
respectively), (C-D) total ion chromatograms for the middle
affibody fragment using Fmoc N-terminal protecting groups and a
traditional manual arrangement using Boc N-terminal protecting
groups, respectively (sequences in FIGS. 8C and 8D correspond to
SEQ ID NOs.: 11 and 12, respectively), and (E-F) total ion
chromatograms for the C-terminal affibody fragment using Fmoc
N-terminal protecting groups and a traditional manual arrangement
using Boc N-terminal protecting groups, respectively (sequences in
FIGS. 8E and 8F correspond to SEQ ID NOs.: 13 and 14,
respectively);
[0020] FIGS. 9A-9E are, according to certain embodiments, (A) a
total ion chromatogram for a purified first affibody fragment (SEQ
ID NO: 6), (B) a total ion chromatogram for a purified second
affibody fragment (SEQ ID NO: 7), (C) a total ion chromatogram for
a purified third affibody fragment (SEQ ID NO: 8), (D) a total ion
chromatogram for the purified affibody fragment from the ligation
of the first and second fragment (SEQ ID NO: 15), and (E) a
chromatogram and mass spectrum for the purified affibody;
[0021] FIG. 10 is a plot of ultraviolet absorbance as a function of
time recorded during the synthesis of a peptide, according to one
set of embodiments;
[0022] FIG. 11 is a graph of flow rate versus wash time, according
to one set of embodiments;
[0023] FIGS. 12A-G are, according to one set of embodiments, (A) a
photograph of an inlet (left) and outlet (right), (B) a photograph
of a spacer, (C) a photograph of a reactor body unit, (D) a
photograph of an assembled reactor, and (E) a schematic of the
reactor showing the reactor body, frit, and spacer, (F) a cutaway
of a reactor, and (G) a synthetic timeline used with a reactor;
[0024] FIG. 13 is a schematic illustration of an exemplary system
for performing peptide synthesis, according to one set of
embodiments;
[0025] FIG. 14 is, according to certain embodiments, an exemplary
total ion chromatogram of a synthesized peptide
ALFALFA-CONHNH.sub.2 (SEQ ID NO: 48);
[0026] FIG. 15 shows two exemplary chromatograms of peptides made
using certain of the peptide synthesis systems described herein
(sequences from left to right correspond to SEQ ID NOs.: 16 to 17,
respectively);
[0027] FIGS. 16A-H are, according to one set on embodiments, crude
LCMS chromatograms for ACP(65-74) (SEQ ID NO: 3) synthesized with a
second generation protocol at (A) 60.degree. C. using HATU, (B)
60.degree. C. using HBTU, (C) room temperature using HBTU, and (D)
room temperature using a comparable manual batch method, and
ACP(65-74) synthesized with a first generation protocol at (E)
60.degree. C. using HATU, (F) 60.degree. C. using HBTU, (G) room
temperature using HBTU, and (H) room temperature using a comparable
manual batch method;
[0028] FIG. 17 includes chromatograms of synthesized biotinylated
peptides, according to some embodiments (sequences from top to
bottom correspond to SEQ ID NOs.: 18 and 19, respectively);
[0029] FIGS. 18A-D are, according to one set of embodiments,
chromatograms of (A) PnIA (A10L) conotoxin (SEQ ID NO: 4)
synthesized with a second generation protocol, (B) HIV-1 PR (81-99)
(SEQ ID NO: 5) synthesized with a second generation protocol, (C)
PnIA (A10L) conotoxin (SEQ ID NO: 4) synthesized with a first
generation protocol, and (D) HIV-1 PR (81-99) (SEQ ID NO: 5)
synthesized with a first generation protocol;
[0030] FIGS. 19A-F are chromatograms, according to certain
embodiments, of (A-C) affibody fragments synthesized on
chlorotrityl hydrazide functionalized polystyrene with a second
generation protocol (sequences in FIGS. 19A-19C correspond to SEQ
ID NOs.: 6, 20, and 8, respectively) and (D-F) affibody fragments
synthesized on modified Wang resin with a first generation protocol
(sequences in FIGS. 19D-19F correspond to SEQ ID NOs.: 6, 7, and 8,
respectively;
[0031] FIG. 20 includes chromatograms of a library of glutathione
analogues synthesized according to one set of embodiments
(sequences from left to right and top to bottom correspond to SEQ
ID NOs.: 21-30, respectively);
[0032] FIGS. 21A-C are chromatograms of cysteine rich peptides
synthesized according to certain embodiments (sequences in FIGS.
21A-21C correspond to SEQ ID NOs.: 31-33, respectively);
[0033] FIGS. 22A-B are chromatograms for (A) (ALF)7 synthesized
using an automated process and (B) (ALF)7 synthesized using a
manual process, according to certain embodiments (sequences in
FIGS. 22A and 22B correspond to SEQ ID NO: 34);
[0034] FIGS. 23A-E are (A) a schematic of an automated flow
platform, (B) a synthetic timeline used by the automated flow
platform to incorporate an amino acid residue, (C) a chromatogram
showing ALFALFA (SEQ ID NO: 48), (D) a chromatogram showing
ACP(65-74) (SEQ ID NO: 3), and (E) a chromatogram showing (ALF)7
(SEQ ID NO: 34), according to one set of embodiments;
[0035] FIGS. 24A-B show (A) a chromatogram of a peptide (SEQ ID NO:
35) synthesized using addition cycles including removal steps and
(B) a chromatogram of a peptide synthesized using addition cycles
lacking one or more removal step;
[0036] FIGS. 25A-F show (A) a synthetic scheme for DARPin (SEQ ID
NO: 36) synthesis, (B-E) chromatograms of fragments DARPin
(sequences in FIGS. 25B to 25E correspond to SEQ ID NOs.: 37-40,
respectively), and (F) a chromatogram of DARPin, according to one
set of embodiments;
[0037] FIGS. 26A-F show (A) a synthetic scheme for Barnase (SEQ ID
NO: 41) synthesis, (B-E) chromatograms of fragments Barnase
(sequences in FIGS. 26B to 26E correspond to SEQ ID NOs.: 42-44 and
35, respectively), and (F) a chromatogram of Barnase, according to
certain embodiments; and
[0038] FIGS. 27A-C show (A) a chromatogram of human insulin A chain
(SEQ ID NO: 45), (B) a chromatogram of human insulin B chain (SEQ
ID NO: 46), and (C) a chromatogram of evolved EETI-II integrin
binding peptide (SEQ ID NO: 47), according to certain
embodiments.
DETAILED DESCRIPTION
[0039] Systems and processes for performing solid phase peptide
synthesis are generally described. Solid phase peptide synthesis is
a known process in which amino acid residues are added to peptides
that have been immobilized on a solid support. In certain
embodiments, the inventive systems and methods can be used to
perform solid phase peptide synthesis quickly while maintaining
high yields. Certain embodiments relate to processes and systems
that may be used to heat, transport, and/or mix reagents in ways
that reduce the amount of time required to perform solid phase
peptide synthesis.
[0040] Certain embodiments relate to a process for adding amino
acid(s) to an immobilized peptide. FIG. 1 is a schematic
illustration of an exemplary system 5 which can be used to perform
certain of the inventive processes described herein. The systems
and methods described herein (e.g. system 5 in FIG. 1) can, in
certain embodiments, involve flow-based synthesis (as opposed to
batch-based synthesis, which is employed in many traditional solid
phase peptide synthesis systems). In some such embodiments,
continuous peptide synthesis can be performed, in which fluid (of
one form or another) is substantially continuously transported over
the immobilized peptides. For example, reagents and rinsing fluids
may be alternatively and continuously transported over the
immobilized peptides, in certain embodiments.
[0041] In some embodiments, peptides 20 may be immobilized on a
solid support 15. Solid support 15 may be contained within a
vessel, such as reactor 10. In some embodiments, and as shown in
FIG. 1, a plurality of reagent reservoirs may be located upstream
of and fluidically connected to reactor 10. In some embodiments, a
reagent reservoir 25 contains amino acids (e.g., pre-activated
amino acids and/or amino acids that are not fully activated). In
some instances, a reagent reservoir 26 contains an amino acid
activating agent (e.g., an alkaline liquid, a carbodiimide, and/or
an uronium activating agent), capable of completing the activation
of the amino acids. In certain embodiments, a reagent reservoir 27
contains a deprotection reagent, such as piperidine or
trifluoroacetic acid. A reagent reservoir 28 may contain a solvent,
such as dimethylformamide (DMF), that may be used, e.g., in a
reagent removal step. While single reservoirs have been illustrated
in FIG. 1 for simplicity, it should be understood that in FIG. 1,
where single reservoirs are illustrated, multiple reservoirs (e.g.,
each containing different types of amino acids, different types of
deprotection agents, etc.) could be used in place of the single
reservoir.
[0042] In certain embodiments, peptides 20 comprise protection
groups, for example, on the N-termini of the peptides. As used
herein, the term "protection group" is given its ordinary meaning
in the art. Protection groups include chemical moieties that are
attached to or are configured to be attached to reactive groups
(i.e., the protected groups) within a molecule (e.g., peptides)
such that the protection groups prevent or otherwise inhibit the
protected groups from reacting. Protection may occur by attaching
the protection group to the molecule. Deprotection may occur when
the protection group is removed from the molecule, for example, by
a chemical transformation which removes the protection group.
[0043] In some embodiments, a plurality of peptides comprising
protection groups may be bound to a solid support such that each
peptide is immobilized on the solid support. For example, the
peptides may be bound to the solid support via their C termini,
thereby immobilizing the peptides.
[0044] In some embodiments, the process of adding amino acid
residues to immobilized peptides comprises exposing a deprotection
reagent to the immobilized peptides to remove at least a portion of
the protection groups from at least a portion of the immobilized
peptides. The deprotection reagent exposure step can be configured,
in certain embodiments, such that side-chain protection groups are
preserved, while N-terminal protection groups are removed. For
instance, in certain embodiments, the protection group used to
protect the peptides comprises fluorenylmethyloxycarbonyl (Fmoc).
In some such embodiments, a deprotection reagent comprising
piperidine (e.g., a piperidine solution) may be exposed to the
immobilized peptides such that the Fmoc protection groups are
removed from at least a portion of the immobilized peptides. In
some embodiments, the protection group used to protect the peptides
comprises tert-butyloxycarbonyl (Boc). In some such embodiments, a
deprotection reagent comprising trifluoroacetic acid may be exposed
to the immobilized peptides such that the Boc protection groups are
removed from at least a portion of the immobilized peptides. In
some instances, the protection groups (e.g., tert-butoxycarbonyl,
i.e., Boc) may be bound to the N-termini of the peptides.
[0045] In some embodiments, the process of adding amino acid
residues to immobilized peptides comprises removing at least a
portion of the deprotection reagent. In some embodiments, at least
a portion of any reaction byproducts (e.g., removed protection
groups) that may have formed during the deprotection step can be
removed. In some instances, the deprotection reagent (and, in
certain embodiments byproducts) may be removed by washing the
peptides, solid support, and/or surrounding areas with a fluid
(e.g., a liquid such as an aqueous or non-aqueous solvent, a
supercritical fluid, and/or the like), for example stored in
optional reservoir 28. In some instances, removing the deprotection
reagent and reaction byproducts may improve the performance of
subsequent steps (e.g., by preventing side reactions). In certain
embodiments, the performance of subsequent steps is not dependent
on the removal of at least a portion (e.g., substantially all) of
the deprotection reagent and/or reaction byproducts. In some such
cases, the removal step is optional. In embodiments in which the
removal step is optional, the removal step may be reduced (e.g.,
reduction in time of the removal step, reduction in the amount of
solvent used in the removal step) and/or eliminated. The reduction
or elimination of one or more removal steps may reduce the overall
cycle time. It should be understood that if an optional removal
step is reduced or eliminated the subsequent step in the addition
cycle may serve to remove at least a portion of the deprotection
reagent and/or reaction byproducts, e.g., due to fluid flow in the
system.
[0046] The process of adding amino acid residues to immobilized
peptides comprises, in certain embodiments, exposing activated
amino acids to the immobilized peptides such that at least a
portion of the activated amino acids are bonded to the immobilized
peptides to form newly-bonded amino acid residues. For example, the
peptides may be exposed to activated amino acids that react with
the deprotected N-termini of the peptides. In certain embodiments,
amino acids can be activated for reaction with the deprotected
peptides by mixing an amino acid-containing stream with an
activation agent stream, as discussed in more detail below. In some
instances, the amine group of the activated amino acid may be
protected, such that addition of the amino acid results in an
immobilized peptide with a protected N-terminus.
[0047] In some embodiments, the process of adding amino acid
residues to immobilized peptides comprises removing at least a
portion of the activated amino acids that do not bond to the
immobilized peptides. In some embodiments, at least a portion of
the reaction byproducts that may form during the activated amino
acid exposure step may be removed. In some instances, the activated
amino acids and byproducts may be removed by washing the peptides,
solid support, and/or surrounding areas with a fluid (e.g., a
liquid such as an aqueous or non-aqueous solvent, a supercritical
fluid, and/or the like), for example stored in optional reservoir
28. In some instances, removing at least a portion of the activated
amino acids and reaction byproducts may improve the performance of
subsequent steps (e.g., by preventing side reactions). In certain
embodiments, the performance of subsequent steps is not dependent
on the removal of at least a portion (e.g., substantially all) of
the activated amino acids and/or reaction byproducts. In some such
cases, the removal step is optional. In embodiments in which the
removal step is optional, the removal step may be reduced (e.g.,
reduction in time of the removal step, reduction in the amount of
solvent used in the removal step) and/or eliminated. The reduction
or elimination of one or more removal step may reduce the overall
cycle time. It should be understood that if an optional removal
step is reduced or eliminated the subsequent step in the addition
cycle may serve to remove at least a portion of the activated amino
acids and/or reaction byproducts, e.g., due to fluid flow in the
system.
[0048] It should be understood that the above-referenced steps are
exemplary and an amino acid addition cycle need not necessarily
comprise all of the above-referenced steps. For example, an amino
acid addition cycle may not include the deprotection reagent
removal step and/or the activated amino acid removal step.
Generally, an amino acid addition cycle includes any series of
steps that results in the addition of an amino acid residue to a
peptide.
[0049] In certain embodiments, during the amino acid addition
steps, adding the amino acid can result in the peptide
incorporating a single additional amino acid residue (i.e., a
single amino acid residue can be added to the immobilized peptides
such that a given peptide includes a single additional amino acid
residue after the addition step). In some such embodiments,
subsequent amino acid addition steps can be used to build peptides
by adding amino acid residues individually until the desired
peptide has been synthesized. In some embodiments, more than one
amino acid residue (e.g., in the form of a peptide) may be added to
a peptide immobilized on a solid support (i.e., a peptide
comprising multiple amino acid residues can be added to a given
immobilized peptide). Addition of peptides to immobilized peptides
can be achieved through processes know to those of ordinary skill
in the art (e.g., fragment condensation, chemical ligation). That
is to say, during the amino acid addition step, adding an amino
acid to an immobilized peptide can comprise adding a single amino
acid residue to an immobilized peptide or adding a plurality of
amino acid residues (e.g., as a peptide) to an immobilized
peptide.
[0050] In some embodiments, amino acids can be added to peptides
significantly faster than conventional methods. In certain
embodiments, the total amount of time taken to perform the
combination of steps may be influenced by the protection group. For
instance, in certain embodiments in which the protection groups
comprise fluorenylmethyloxycarbonyl (Fmoc), the total amount of
time taken to perform the combination of all of the deprotection
reagent exposing step, the deprotection reagent removal step, the
activated amino acid exposing step, and the activated amino acid
removal step is about 10 minutes or less, about 9 minutes or less,
about 8 minutes or less, about 7 minutes or less, about 6 minutes
or less, about 5 minutes or less, about 4 minutes or less, about 3
minutes or less, about 2 minutes or less, about 1 minute or less,
from about 10 seconds to about 10 minutes, from about 10 seconds to
about 9 minutes, from about 10 seconds to about 8 minutes, from
about 10 seconds to about 7 minutes, from about 10 seconds to about
6 minutes, from about 10 seconds to about 5 minutes, from about 10
seconds to about 4 minutes, from about 10 seconds to about 3
minutes, from about 10 seconds to about 2 minutes, or from about 10
seconds to about 1 minute. In certain embodiments (including
embodiments in which the protection groups comprise
tert-butyloxycarbonyl (Boc), fluorenylmethyloxycarbonyl (Fmoc),
and/or other types of protection groups), the total amount of time
taken to perform the combination of all of the deprotection reagent
exposing step, the deprotection reagent removal step, the activated
amino acid exposing step, and the activated amino acid removal step
is about 5 minutes or less, about 4 minutes or less, about 3
minutes or less, about 2 minutes or less, about 1 minute or less,
from about 10 seconds to about 5 minutes, from about 10 seconds to
about 4 minutes, from about 10 seconds to about 3 minutes, from
about 10 seconds to about 2 minutes, or from about 10 seconds to
about 1 minute.
[0051] In general, the total amount of time taken to perform the
combination of all of the deprotection reagent exposing step, the
deprotection reagent removal step, the activated amino acid
exposing step, and the activated amino acid removal step is
calculated by adding the amount of time it takes to perform the
deprotection reagent exposing step to the amount of time it takes
to perform the deprotection reagent removal step and to the amount
of time it takes to perform the activated amino acid exposing step
and to the amount of time it takes to perform the activated amino
acid removal step.
[0052] In embodiments in which an addition cycle lacks one or more
removal steps, as described herein, and the protection groups
comprise fluorenylmethyloxycarbonyl (Fmoc), the total amount of
time taken to perform the combination of all of the steps of the
addition cycle (e.g., the deprotection reagent exposing step, the
activated amino acid exposing step, and the activated amino acid
removal step; the deprotection reagent exposing step, the
deprotection reagent removal step, and the activated amino acid
exposing step; the deprotection reagent exposing step and the
activated amino acid exposing step) is about 10 minutes or less,
about 9 minutes or less, about 8 minutes or less, about 7 minutes
or less, about 6 minutes or less, about 5 minutes or less, about 4
minutes or less, about 3 minutes or less, about 2 minutes or less,
about 1 minute or less, about 0.75 minute or less, from about 10
seconds to about 10 minutes, from about 10 seconds to about 9
minutes, from about 10 seconds to about 8 minutes, from about 10
seconds to about 7 minutes, from about 10 seconds to about 6
minutes, from about 10 seconds to about 5 minutes, from about 10
seconds to about 4 minutes, from about 10 seconds to about 3
minutes, from about 10 seconds to about 2 minutes, or from about 10
seconds to about 1 minute. In certain embodiments (including
embodiments in which the protection groups comprise
tert-butyloxycarbonyl (Boc),fluorenylmethyloxycarbonyl (Fmoc),
and/or other types of protection groups), the total amount of time
taken to perform the combination of all of the steps in an addition
cycle lacking one or more removal steps is about 5 minutes or less,
about 4 minutes or less, about 3 minutes or less, about 2 minutes
or less, about 1 minute or less, from about 10 seconds to about 5
minutes, from about 10 seconds to about 4 minutes, from about 10
seconds to about 3 minutes, from about 10 seconds to about 2
minutes, or from about 10 seconds to about 1 minute.
[0053] In general, the total amount of time taken to perform the
combination of all of the steps in an addition cycle lacking one or
more removal steps is calculated by adding the amount of time it
takes to perform the deprotection reagent exposing step to the
amount of time it takes to perform the deprotection reagent removal
step, if present, and to the amount of time it takes to perform the
activated amino acid exposing step and to the amount of time it
takes to perform the activated amino acid removal step, if
present.
[0054] In certain embodiments, the first amino acid addition step
(and/or subsequent amino acid addition steps) may add amino acids
at a relatively high yield. For example, certain embodiments
include exposing amino acids to the immobilized peptides such that
an amino acid residue is added to at least about 99%, at least
about 99.9%, at least about 99.99%, or substantially 100% of the
immobilized peptides. In certain embodiments, a second (and, in
some embodiments, a third, a fourth, a fifth, and/or a subsequent)
amino acid addition cycle can be performed which may include
exposing amino acids to the immobilized peptides such that an amino
acid residue is added to at least about 99%, at least about 99.9%,
at least about 99.99%, or substantially 100% of the immobilized
peptides. In certain embodiments, the use of processes and systems
of the present invention may allow the addition of more than one
amino acid to the immobilized peptides to occur relatively quickly
(including within any of the time ranges disclosed above or
elsewhere herein), while maintaining a high reaction yield.
[0055] In certain embodiments, one or more amino acid addition
steps can be performed while little or no double incorporation
(i.e., adding multiple copies of a desired amino acid (e.g., single
amino acid residues or peptides) during a single addition step).
For example, in certain embodiments, multiple copies of the desired
amino acid are bonded to fewer than about 1% (or fewer than about
0.1%, fewer than about 0.01%, fewer than about 0.001%, fewer than
about 0.0001%, fewer than about 0.00001%, or substantially none) of
the immobilized peptides during a first (and/or second, third,
fourth, fifth, and/or subsequent) amino acid addition step.
[0056] In some embodiments, multiple amino acid addition cycles can
be performed. Performing multiple amino acid addition cycles can
result in more than one single-amino-acid residue (or more than one
peptide, and/or at least one single-amino-acid residue and at least
one peptide) being added to a peptide. In certain embodiments a
process for adding more than one amino acid to immobilized peptides
may comprise performing a first amino acid addition cycle to add a
first amino acid and performing a second amino acid addition cycle
to add a second amino acid. In certain embodiments, third, fourth,
fifth, and subsequent amino acid addition cycles may be performed
to produce an immobilized peptide of any desired length. In some
embodiments, at least about 10 amino acid addition cycles, at least
about 50 amino acid addition cycles, or at least about 100 amino
acid addition cycles are performed, resulting in the addition of at
least about 10 amino acid residues, at least about 50 amino acid
residues, or at least about 100 amino acid residues to the
immobilized peptides. In certain such embodiments, a relatively
high percentage of the amino acid addition cycles (e.g., at least
about 50%, at least about 75%, at least about 90%, at least about
95%, or at least about 99% of such amino acid addition cycles) can
be performed at high yield (e.g., at least about 99%, at least
about 99.9%, at least about 99.99%, or substantially 100%). In some
such embodiments, a relatively high percentage of the amino acid
addition cycles (e.g., at least about 50%, at least about 75%, at
least about 90%, at least about 95%, or at least about 99% of such
amino acid addition cycles) can be performed quickly, for example,
within any of the time ranges specified above or elsewhere herein.
In some such embodiments, a relatively high percentage of the amino
acid addition cycles (e.g., at least about 50%, at least about 75%,
at least about 90%, at least about 95%, or at least about 99% of
such amino acid addition cycles) can be performed with limited or
no double incorporation, for example, within any of the double
incorporation ranges specified above or elsewhere herein.
[0057] In embodiments in which there are more than one addition
cycles, the total amount of time that passes between the end of an
amino acid addition cycle and a subsequent amino acid addition
cycle may be relatively short. For example, in certain embodiments
in which fluorenylmethyloxycarbonyl protection groups are employed,
the total amount of time between the ends of the first and second
amino acid addition cycles is about 10 minutes or less, about 9
minutes or less, about 8 minutes or less, about 7 minutes or less,
about 6 minutes or less, about 5 minutes or less, about 4 minutes
or less, about 3 minutes or less, about 2 minutes or less, about 1
minute or less, from about 10 seconds to about 10 minutes, from
about 10 seconds to about 9 minutes, from about 10 seconds to about
8 minutes, from about 10 seconds to about 7 minutes, from about 10
seconds to about 6 minutes, from about 10 seconds to about 5
minutes, from about 10 seconds to about 4 minutes, from about 10
seconds to about 3 minutes, from about 10 seconds to about 2
minutes, or from about 10 seconds to about 1 minute. In certain
embodiments in which protection groups comprising
fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, and/or any other
suitable protection group are employed, the total amount of time
between the end of an amino acid addition cycle and a subsequent
amino acid addition cycle may be about 5 minutes or less, about 4
minutes or less, about 3 minutes or less, about 2 minutes or less,
about 1 minute or less, from about 10 seconds to about 5 minutes,
from about 10 seconds to about 4 minutes, from about 10 seconds to
about 3 minutes, from about 10 seconds to about 2 minutes, or from
about 10 seconds to about 1 minute.
[0058] As mentioned above, certain aspects relate to processes and
systems that allow the total time required for one or more addition
cycles to be significantly reduced compared to previous solid phase
peptide synthesis methods. Since the advent of continuous solid
phase peptide synthesis over 30 years ago, continual efforts have
focused on improving its utility and applicability. While these
improvements have contributed to the commercial success of
automated solid phase peptide synthesizers, reducing synthesis time
still remains a significant barrier. Over 30 years of research and
development in the field have been unable to produce fast synthesis
techniques. Typical continuous solid phase peptide synthesis using
Fmoc or Boc protection groups require 30 to 90 minutes to add a
single amino acid. Certain processes and techniques have been
discovered, with the context of the present invention, that address
the long felt need to decrease synthesis time. For example, fast
synthesis times may be achieved by employing specialized techniques
for mixing, heating, and/or controlling pressure drop.
[0059] Certain steps in the amino acid addition cycle may require
mixing of reagents. In some conventional systems, reagents are
mixed a long time before exposure to the immobilized peptides,
which may result in undesirable side reactions and/or reagent
degradation prior to exposure to the immobilized peptides. In some
instances, the side reactions and/or degradation adversely affects
the yield and kinetics of one or more steps in the amino acid
addition cycle (e.g., amino acid exposing step). In some
conventional systems, reagents are mixed in the presence of the
immobilized peptides, which may result, e.g., in slower reaction
kinetics. One technique for achieving rapid peptide synthesis may
involve merging reagent streams prior to, but within a short amount
of time of, arrival at the immobilized peptides, as shown in FIG.
1.
[0060] In some embodiments, a process for adding amino acid
residues to peptides comprises flowing a first stream comprising
amino acids, flowing a second stream comprising an amino acid
activating agent (e.g., an alkaline liquid, a carbodiimide, and/or
an uronium activating agent). For example, referring back to FIG.
1, reagent reservoir 25 may comprise amino acids. Reagent reservoir
26 may comprise, in some such embodiments, an amino acid activating
agent. The first and second streams may be merged to form a mixed
fluid comprising activated amino acids. For example, referring to
FIG. 1, amino acids from reservoir 25 can be flowed in first stream
30, and amino acid activating agent can be flowed in second stream
32. First stream 30 and second stream 32 can be mixed, for example,
at point 34 of stream 40. The mixed fluid may comprise activated
amino acids due to the activation of the amino acids by the amino
acid activating agent.
[0061] In certain embodiments, after the amino acids have been
activated, the immobilized peptides may be exposed to the mixed
fluid within a relatively short period of time. For example, in
certain embodiments, the plurality of peptides immobilized on the
solid support may be exposed to the mixed fluid within about 30
seconds (or within about 15 seconds, within about 10 seconds,
within about 5 seconds, within about 3 seconds, within about 2
seconds, within about 1 second, within about 0.1 seconds, or within
about 0.01 seconds) after merging the first and second streams to
form the mixed fluid.
[0062] In certain embodiments, merging reagent streams may be used
in an amino acid addition cycle, as described herein. For example,
a first fluid stream comprising amino acids and a second stream
comprising an amino acid activating agent may be merged to form a
mixed fluid comprising the activated amino acids within about 30
seconds prior to exposing the activated amino acids to peptides
immobilized on a solid support. In some embodiments, in which more
than one amino acid addition cycle is performed, one or more amino
acid addition cycles (e.g., a first and a second amino acid
addition cycle) may comprise merging a first fluid stream
comprising amino acids and a second stream comprising an amino acid
activating agent to form a mixed fluid comprising activated amino
acids within about 30 seconds prior to exposing the amino acids to
the solid support. It should be understood that merging reagent
streams may be used in connection with any suitable step in the
addition cycle and may be used in connection with one or more steps
in an amino acid addition cycle.
[0063] In general, streams may be merged using any suitable
technique known to those of skill in the art. In some embodiments,
the streams may be merged by flowing the first stream and the
second stream substantially simultaneously into a single stream
(e.g., by merging channels through which the streams flow). Other
merging methods may also be used.
[0064] Another technique for achieving fast synthesis times may
involve heating a stream prior to, but within a short period of
time of, arrival at the reactor. Supplying the reactor with a
heated stream may alter the kinetics of a process occurring in the
reactor. For example, exposing immobilized peptides, solid
supports, or other synthesis components to a heated stream may
alter the reaction kinetics and/or diffusion kinetics of the amino
acid addition process. For example, exposing the peptides to a
heated stream comprising activated amino acids may increase the
rate at which amino acids are added to the peptides. In some
embodiments, heating the stream prior to, but within a short period
of time of arrival at the reactor may substantially reduce or
eliminate the need to supply auxiliary heat (i.e., heat that is not
from one or more pre-heated streams) to the reactor. In some
instances, most or substantially all of the heat supplied to the
reactor originates from the pre-heated stream. For example, in some
embodiments, the percentage of thermal energy that is used to heat
the reactor that originates from the pre-heated stream(s) may be
greater than or equal to about 50%, greater than or equal to about
60%, greater than or equal to about 70%, greater than or equal to
about 80%, greater than or equal to about 90%, greater than or
equal to about 95%, or greater than or equal to about 99%. In some
such embodiments, heating the system in this way can reduce the
time required to heat the reactor, immobilized peptides, solid
support, activated amino acids, deprotection reagents, wash fluids,
and/or other synthesis components to a desirable reaction
temperature.
[0065] Thus, in some embodiments, a process for adding amino acid
residues to peptides may comprise heating a stream comprising
activated amino acids such that the temperature of the activated
amino acids is increased by at least about 1.degree. C. (or at
least about 2.degree. C., at least about 5.degree. C., at least
about 10.degree. C., at least about 25.degree. C., at least about
50.degree. C., and/or less than or equal to about 450.degree. C.,
less than or equal to about 300.degree. C., less than or equal to
about 200.degree. C., less than or equal to about 100.degree. C.,
and/or less than or equal to about 75.degree. C.) prior to the
heated amino acids being exposed to the immobilized peptides. In
certain embodiments, a stream comprising any other component (e.g.,
a washing agent, a deprotection agent, or any other components) may
be heated such that the temperature of the stream contents is
increased by at least about 1.degree. C. (or at least about
2.degree. C., at least about 5.degree. C., at least about
10.degree. C., at least about 25.degree. C., at least about
50.degree. C., and/or less than or equal to about 450.degree. C.,
less than or equal to about 300.degree. C., less than or equal to
about 200.degree. C., less than or equal to about 100.degree. C.,
and/or less than or equal to about 75.degree. C.) prior to the
stream contents being exposed to the immobilized peptides. In some
instances, the heating step (e.g., the heating of the activated
amino acids and/or the heating of any other component within a
stream transported to the immobilized peptides) may be performed
within about 30 seconds (or within about 15 seconds, within about
10 seconds, within about 5 seconds, within about 3 seconds, within
about 2 seconds, within about 1 second, within about 0.1 seconds,
or within about 0.01 seconds) of exposing the stream contents
(e.g., the heated activated amino acids) to the immobilized
peptides. In some such embodiments, and as illustrated in the
exemplary embodiment of FIG. 1, such heating may be achieved by
heating a location upstream of the immobilized peptides. In some
such embodiments, the heating of the amino acids begins at least
about 0.01 seconds, at least about 0.05 seconds, at least about 0.1
seconds, at least about 0.5 seconds, at least about 1 second, at
least about 5 seconds, or at least about 10 seconds prior to
exposure of the amino acids to the immobilized peptides. In certain
embodiments, the amino acids are heated by at least about 1.degree.
C. (or at least about 2.degree. C., at least about 5.degree. C., at
least about 10.degree. C., at least about 25.degree. C., at least
about 50.degree. C., and/or less than or equal to about 450.degree.
C., less than or equal to about 300.degree. C., less than or equal
to about 200.degree. C., less than or equal to about 100.degree.
C., and/or less than or equal to about 75.degree. C.) at least
about 0.1 seconds, at least about 1 second, at least about 5
seconds, or at least about 10 seconds prior to the amino acids
being exposed to the immobilized peptides.
[0066] Referring back to FIG. 1, for example, system 5 may comprise
heating zone 42, within which the contents of stream 40 may be
heated. Heating zone 42 may comprise a heater. In general, any
suitable method of heating may be used to increase the temperature
of a stream. One advantage of certain of the systems and methods
described herein, is the ability to use simple and/or inexpensive
heating methods and/or apparatus. For example, heating zone 42 may
comprise a liquid bath (e.g., a water bath), a resistive heater, a
gas convection-based heating element, or any other suitable heater.
In some embodiments, a relatively low percentage of the thermal
energy used to heat the inlet stream (and/or the reactor) may
originate from electromagnetic radiation (e.g., microwave
radiation). For example, in some embodiments, the percentage of the
thermal energy used to heat the inlet stream(s) and/or the reactor
that originates from electromagnetic radiation may be less than or
equal to about 20%, less than or equal to about 15%, less than or
equal to about 10%, less than or equal to about 5%, less than or
equal to about 1%, or less than or equal to about 0.5%. In some
embodiments, inlet stream(s) and/or the reactor may be heated
without the use of electromagnetic radiation. In certain
embodiments, the percentage of the thermal energy used to heat the
inlet stream(s) and/or the reactor that originates from microwave
radiation may be less than or equal to about 20%, less than or
equal to about 15%, less than or equal to about 10%, less than or
equal to about 5%, less than or equal to about 1%, or less than or
equal to about 0.5%. In some embodiments, inlet stream(s) and/or
the reactor may be heated without the use of microwave radiation.
In some instances, the heating mechanism may be within a short
distance of the immobilized peptides, for example, within about 5
meters, within about 1 meter, within about 50 cm, or within about
10 cm.
[0067] In some embodiments, including those illustrated in FIG. 1,
both the heating of the amino acids and the merging of the amino
acids with the amino acid activating agent (e.g., an alkaline
liquid, a carbodiimide, and/or a uronium activating agent) can be
performed before and within a relatively short time of the amino
acids contacting the immobilized peptides. Heating the amino acids
may be performed before, during, and/or after merging the stream
comprising the amino acids with the stream comprising the amino
acid activating agent.
[0068] In certain embodiments, heating a stream just prior to being
exposed to the immobilized peptides (as opposed to heating the
stream long before transport of the stream contents to the
immobilized peptides) may minimize the thermal degradation of one
or more reagents (such as, for example, the amino acids that are to
be added to the peptides and/or the deprotection reagent) in the
stream. Of course, as discussed above, heating a stream prior to
arrival of the stream components can enhance the speed with which a
reaction or washing step may be performed.
[0069] In some embodiments, a heating step may be used in an amino
acid addition cycle, as described herein. For example, heating the
activated amino acids, such that the temperature of the activated
amino acids is increased by at least about 1.degree. C., may be
performed prior to and within about 30 seconds (or within any of
the other time ranges mentioned elsewhere) of exposing the
activated amino acids to the immobilized peptides. It should be
understood that a heating step may be used prior to and within 30
seconds of any step of an amino acid addition cycle (e.g.,
deprotection reagent exposing step, deprotection reagent removal
step, activated amino acid exposing step, activated amino acid
removal step). In some embodiments, an amino acid addition cycle
may comprise more than one heating step. For example, a heating
step may be performed before a deprotection reagent exposing step
and an activated amino acid exposing step.
[0070] In certain embodiments, in which more than one amino acid
addition cycle is performed, one or more amino acid addition cycles
(e.g., a first and a second amino acid addition cycle) may comprise
one or more heating steps prior to and within about 30 seconds (or
within any of the other time ranges mentioned elsewhere) of
performing a step (e.g. deprotection reagent exposing step,
deprotection reagent removal step, activated amino acid exposing
step, activated amino acid removal step). For example, one or more
amino acid addition cycles (e.g., a first and a second amino acid
addition cycle) may comprise heating the activated amino acids
prior to and within about 30 seconds (or within any of the other
time ranges mentioned elsewhere) of exposing the activated amino
acids to the immobilized peptides. In general, a heating step may
be used in connection with any suitable step in the addition cycle
and may be used in connection with one or more steps of any
individual addition cycle or with all steps of a series of addition
cycles.
[0071] As noted above, in some embodiments, heating a stream may
increase the temperature of the stream contents (e.g., may increase
the temperature of amino acids within the stream) by at least about
1.degree. C., at least about 2.degree. C., at least about 5.degree.
C., at least about 10.degree. C., at least about 25.degree. C., or
at least about 50.degree. C. It should be understood that the
temperature of the stream after heating may be the same or
different for different addition cycle steps and/or addition
cycles. In some instances, the temperature of the stream after
heating may be the same for one or more addition cycle steps and/or
addition cycles. In some instances, heating a stream may increase
the temperature of the stream contents (e.g., may increase the
temperature of amino acids within the stream) by less than or equal
to about 450.degree. C., less than or equal to about 300.degree.
C., less than or equal to about 200.degree. C., less than or equal
to about 100.degree. C. or less than or equal to about 75.degree.
C. Combinations of the above-referenced ranges are also possible
(e.g., at least about 1.degree. C. and less than or equal to about
100.degree. C., at least about 1.degree. C. and less than or equal
to about 450.degree. C., etc.).
[0072] Systems and methods for reducing pressure drop across the
immobilized peptides may be used, according to certain embodiments,
to improve the speed of peptide synthesis. In some embodiments, the
flow rate of reagents across the immobilized peptides may influence
the speed of peptide synthesis. For example, the time required for
one or more steps in an amino acid addition cycle (e.g.,
deprotection reagent exposing step, deprotection reagent removal
step, activated amino acid exposing step, activated amino acid
removal step) may decrease with increasing flow rate. In general,
the use of high flow rates ensures that the concentration of
reagent near the immobilized peptides is not depleted as severely
as might be observed when low flow rates are employed. In many
traditional continuous solid phase peptide synthesis systems, the
flow rate is limited by the pressure drop across the reactor.
Pressure drop may occur due to expansion of the solid support
during synthesis and/or due to improper sizing of process
equipment. In certain embodiments, the pressure drop across the
solid support during an amino acid addition cycle may not exceed
about 700 psi for more than about 5% (or for more than about 1%) of
the time period during which the cycle is performed. For example,
in certain embodiments, during each step of an amino acid addition
cycle (e.g. the deprotection reagent exposing step, the
deprotection reagent removal step, the activated amino acid
exposing step, and the activated amino acid removal step) the
pressure drop across the solid support may not exceed about 700 psi
for more than about 5% (or for more than about 1%) of the time
period during which the step is performed. In embodiments in which
more than one addition cycle is performed, the pressure drop during
one or more addition cycles (e.g., the first and second amino acid
addition cycle) may not exceed about 700 psi for more than about 5%
(or for more than about 1%) of the time period during which the
cycle is performed.
[0073] In some embodiments, the pressure drop across reactor during
each step of an amino acid addition cycle and/or during one or more
addition cycles may not exceed about 700 psi, about 600 psi, about
500 psi, about 400 psi, about 250 psi, about 100 psi, or about 50
psi for more than about 5% (or for more than about 1%) of the time
period during which the step is performed.
[0074] In certain embodiments, the pressure drop across reactor may
be reduced by using a process vessel (e.g., the column of a packed
column) with a desirable aspect ratio. Generally, the aspect ratio
of a process vessel is the ratio of the length of the vessel
(substantially parallel to the direction of flow through the
vessel) to the shortest width of the vessel (measured perpendicular
to the length of the vessel). As an example, in the case of a
cylindrical vessel, the aspect ratio would be the ratio of the
height of the cylinder to the cross-sectional diameter of the
cylinder. Referring back to FIG. 1, for example, the aspect ratio
of reactor 10 would be the ratio of the length of dimension A to
the length of dimension B (i.e., A:B). In some embodiments, the
aspect ratio of the reactor may be less than or equal to about
20:1, less than or equal to about 10:1, less than or equal to about
5:1, less than or equal to about 3:1, less than or equal to about
2:1, less than or equal to about 1:1, less than or equal to about
0.5:1, less than or equal to about 0.2:1, or less than or equal to
about 0.1:1 (and/or, in certain embodiments, as low as 0.01:1, or
lower).
[0075] In some embodiments, relatively short addition cycles with
high yields and/or limited and/or no double incorporation may be
achieved by employing one or more of the techniques described
herein. For example, certain of the systems and methods described
herein may allow the amino acid exposing step (i.e., the step of
exposing the activated amino acids to the immobilized peptides) to
be performed (e.g., while achieving the high yields and/or avoiding
double incorporation to any of the degrees described herein) in
about 1 minute or less (e.g., about 30 seconds or less, about 15
seconds or less, about 10 seconds or less, about 7 seconds or less,
or about 5 seconds or less, and/or, in certain embodiments, in as
little as 1 second, or less). In some instances, certain of the
systems and methods described herein may allow the deprotection
reagent removal step and/or the activated amino acid removal step
to be performed in about 2 minutes or less (e.g., about 1.5 minutes
or less, about 1 minute or less, about 45 seconds or less, about 30
seconds or less, about 15 seconds or less, about 10 seconds or
less, about 5 seconds or less, and/or, in certain embodiments, in
as little as 1 second, or less). In certain embodiments, certain of
the systems and methods described herein may allow the deprotection
reagent exposing step (i.e., the step of exposing the immobilized
peptides to the deprotection reagent) to be performed in about 20
seconds or less (e.g., about 15 seconds or less, about 10 seconds
or less, about 8 seconds or less, about 5 seconds or less, about 1
second or less, and/or, in certain embodiments, in as little as 0.5
seconds, or less).
[0076] In certain cases, the time required for peptide synthesis
may be influenced by the choice of protection group. For example,
the use of Fmoc protection groups is generally understood to
require longer synthesis cycle times. However, the systems and
methods described herein can be used to perform fast amino acid
addition, even when Fmoc protection group chemistries are employed.
In some embodiments, the total time for an amino acid addition
cycle may be low, regardless of the type of protection group that
is being used.
[0077] In general, any protection group known to those of ordinary
skill in the art can be used. Non-limiting examples of protection
groups (e.g., n-terminal protection groups) include
fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, allyloxycarbonyl
(alloc), carboxybenzyl, and photolabile protection groups. In
certain embodiments, immobilized peptides comprise
fluorenylmethyloxycarbonyl protection groups. In some embodiments,
immobilized peptides comprise tert-butyloxycarbonyl protection
groups.
[0078] As described elsewhere, an amino acid activating agent may
be used to activate or complete the activation of amino acids prior
to exposing the amino acids to immobilized peptides. Any suitable
amino acid activating agent may be used. In certain embodiments,
the amino acid activating agent comprises an alkaline liquid. The
amino acid activating agent comprises, in some embodiments, a
carbodiimide, such as N,N'-dicyclohexylcarbodiimide (DCC),
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the like.
In certain embodiments, the amino acid activating agent comprises a
uronium activating agent, such as
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
hexafluorophosphate (HBTU);
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU);
1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)
dimethylaminomorpholino)] uronium hexafluorophosphate (COMU); and
the like.
[0079] As described elsewhere, peptides may be immobilized on a
solid support. In general, any solid support may be used with any
of the addition cycles described herein. Non-limiting examples of
solid support materials include polystyrene (e.g., in resin form
such as microporous polystyrene resin, mesoporous polystyrene
resin, macroporous polystyrene resin), glass, polysaccharides
(e.g., cellulose, agarose), polyacrylamide resins, polyethylene
glycol, or copolymer resins (e.g., comprising polyethylene glycol,
polystyrene, etc.).
[0080] The solid support may have any suitable form factor. For
example, the solid support can be in the form of beads, particles,
fibers, or in any other suitable form factor.
[0081] In some embodiments, the solid support may be porous. For
example, in some embodiments macroporous materials (e.g.,
macroporous polystyrene resins), mesoporous materials, and/or
microporous materials (e.g., microporous polystyrene resin) may be
employed as a solid support. The terms "macroporous," "mesoporous,"
and "microporous," when used in relation to solid supports for
peptide synthesis, are known to those of ordinary skill in the art
and are used herein in consistent fashion with their description in
the International Union of Pure and Applied Chemistry (IUPAC)
Compendium of Chemical Terminology, Version 2.3.2, Aug. 19, 2012
(informally known as the "Gold Book"). Generally, microporous
materials include those having pores with cross-sectional diameters
of less than about 2 nanometers. Mesoporous materials include those
having pores with cross-sectional diameters of from about 2
nanometers to about 50 nanometers. Macroporous materials include
those having pores with cross-sectional diameters of greater than
about 50 nanometers and as large as 1 micrometer.
[0082] One advantage of the inventive systems and methods described
herein is that they can be used with standard solid support
materials without degradation in performance. For example, in
certain embodiments, a standard commercial polystyrene resin
support can be used. In many previous systems, such supports
collapsed when used in flow-based solid phase peptide synthesis
systems, causing an increase in pressure drop. As the resin swells
during synthesis, it becomes increasingly likely to collapse, which
causes an increase in the pressure drop across the resin, requiring
an increase in applied pressure to maintain a constant flow rate.
The increase in applied pressure can lead to more severe collapse
of the resin, leading to a positive feedback effect in which the
pressure applied to the fluid must be repeatedly increased. At
sufficiently high pressures, the resin may extrude through any frit
or other system used to confine it. The systems and methods
described herein can be used to manage pressure drop such that the
resin (including standard polystyrene resins and other standard
resins) do not collapse during synthesis or collapse only to a
degree that does not result in the positive feedback effect
described above, leading to a more stable and controllable system.
In certain embodiments, the solid support is contained within a
packed column.
[0083] In general, any peptide and/or protein may be synthesized
using the methods and systems described herein. Non-limiting
examples of peptides and/or proteins that may be synthesized using
the methods and/or systems, described herein, include Glucagon-like
peptides (e.g., GLP-1), Exenatide, Liraglutide, GLP-1 analogs
(e.g., ZP10), Pramlinitide, Peptide YY, Glucagon, Teduglutide,
Delmitide, Calcitonin (e.g., Salmon Calcitonin), parathyroid
hormone, Bortezomib, Cilengitide, Leuprorelin, Histrelin,
Goserelin, Stimuvax, Primovax, Nesiritide, Eptifibatide,
Bivalirudin, Icatibant, Rotigaptide, Cyclosporin, MPB8298,
Octreotide, Lanreotide, Desmopressin, Lypressin, Terlipressin,
Oxytocin, Atosiban, Enfuvirtide, Thymalfasin, Daptamycin, Dentonin,
Bacitracin, Gramidicin, Colistin, Pexiganan, Omiganan, Caspofungin,
Micafungin, Anidulafungin, Histatin, Lactoferrin, Conotoxin,
Nemifitide, natriuretic peptide, Vasopressin, Vasopressin analogs
(e.g., Arg vasopressin, Lys vasopressin), Ziconotide,
Echinocandins, Thymalfasin, and Somatostatin analogs (e.g.,
Octreotide, Lanreotide). Peptides and/or proteins for the treatment
of diseases, such as, but not limited to, diabetes,
gastroenterological disorders, orthopedic disorders (e.g.,
osteoporosis), cancer, cardiovascular disease, immunological
disorders (i.e., autoimmune disorders), acromegaly, enuresis,
infections (e.g., bacterial infections, fungal infections, viral
infections), and central nervous system disorders, may be
synthesized using the methods and systems described herein.
[0084] As used herein, the term "peptide" has its ordinary meaning
in the art and may refer to amides derived from two or more amino
carboxylic acid molecules (the same or different) by formation of a
covalent bond from the carbonyl carbon of one to the nitrogen atom
of another with formal loss of water. An "amino acid residue" also
has its ordinary meaning in the art and refers to the composition
of an amino acid (either as a single amino acid or as part of a
peptide) after it has combined with a peptide, another amino acid,
or an amino acid residue. Generally, when an amino acid combines
with another amino acid or amino acid residue, water is removed,
and what remains of the amino acid is called an amino acid residue.
The term "amino acid" also has its ordinary meaning in the art and
may include proteogenic and non-proteogenic amino acids.
[0085] The following examples are intended to illustrate certain
embodiments of the present invention, but do not exemplify the full
scope of the invention.
EXAMPLES
[0086] Standard solid phase peptide synthesis methods utilizing,
e.g., Fmoc as a protecting group may require about 60 to 100
minutes to incorporate each amino acid residue and some specialized
procedures use complex microwave systems to reduce this to about 20
minutes per residue. These examples describe the development of a
flow platform that incorporates an amino acid residue in less than
10 minutes (e.g., every five minutes, every three minutes, every
two minutes) under manual control or 1.8 minutes under automatic
control, without microwave irradiation.
[0087] The flow based platform, described herein, further
accelerates SPPS chemistry, far beyond what is believed to be
currently possible with microwave assisted or other rapid peptide
synthesizers, by leveraging a flow based approach. In addition to
constantly supplying high concentration reagents, the flow-based
platform overcomes a number of significant obstacles that can
hinder standard and microwave-assisted approaches. First, the
completely sealed reactor and heat exchanger can be immersed in a
temperature controlled bath which allows solvents and reagents to
be heated in a consistent and controlled manner immediately before
reaching the resin bed. Rapid preheating is, according to certain
embodiments, important to avoid thermal degradation of reagents
while quickly reaching the desired temperature. However, this is
extremely difficult in a batch system. Second, the flow platform
can be scaled without increasing the cycle times. As demonstrated
in the transition from the first to second generation reactors,
increasing the diameter and flow rate effectively increases the
maximum scale, without slowing the synthesis. Third, stirring is
not required to effect adequate mass transfer, eliminating
failure-prone moving parts, and facilitating scale up. Fourth, high
quality peptides can be obtained quickly without double coupling,
double deprotection, or colorimetric tests of coupling efficiency.
Finally, automation of this system can allow for faster cycle
times, in contrast to the often slow automation of batch
synthesis.
Example 1
[0088] This example describes a flow based platform for rapid Fmoc
solid phase peptide synthesis, in which each amino acid addition
cycle was completed in less than five minutes. In this example,
each step for amino acid addition (e.g., amide bond formation,
washes, and N-termini deprotection) was carried out under a
constant stream of fluid passed over a resin confined in a small,
fritted plastic tube. Flow methods, as opposed to commonly used
batch methods, allowed for the consistent rapid preheating,
addition, and removal of solvents and reagents. The consistent
rapid preheating, addition, and removal of solvents and reagents
allowed a 5 minute cycle time, which included a 30 second
amide-bond formation step. A number of model peptides were
prepared, without double coupling or double deprotection. In
addition, good yields and high purity, as shown by liquid
chromatography-mass spectrometry (LC-MS), were achieved. This
approach was also applied to the synthesis of a 58-residue protein
from three polypeptide segments. The longest fragment, a 27 residue
peptide, was prepared in 2.3 hours, which was 10 fold faster than
conventional Fmoc methods. It is believed that automating various
processing steps, increasing flow rate, reducing unnecessarily long
wash times, and using a smaller aspect ratio reactor would
substantially reduce the synthesis times reported here.
[0089] As shown in FIG. 2A, a high pressure liquid chromatography
(HPLC) pump was used to deliver either a piperidine deprotection
solution or a dimethylformamide (DMF) wash solvent to the reactor.
A manually actuated 3-way valve was used to select which reagent
was delivered to the reactor. The HPLC pump outlet was attached to
the reactor via a luerlock quick connect. There was eight feet of
1/16'' OD.times.0.03'' ID tubing between the luerlock quick connect
and reactor that served as a heat exchanger, or "preheat loop". For
the coupling step, the quick connect was manually moved to a
syringe pump, which delivered a solution of activated amino acid.
It is believed that even faster performance than that reported here
could be achieved by automating this step. The reactor effluent was
passed through a UV detector to continuously monitor the absorbance
at 304 nm, a region where Fmoc amino acids absorb strongly. The
reactor was designed to be simple and easy to construct. A 1/4''
inner diameter by 3.5'' long perfluoroalkoxy tube with Swagelok
reducing unions as the inlet and outlet was used. A frit was
positioned in the outlet using a short piece of tubing with a 1/4
in. outer diameter. Installation of the outlet fitting and
concurrent compression of the ferrule and tube sealed the frit in
place as seen in FIG. 2B. The total volume of the reactor was about
2.5 ml. This design held up to 100 mg of resin and was used to
prepare peptides up to 27 residues in length.
[0090] To verify the feasibility of Fmoc SPPS with this flow based
SPPS system, the model peptide Fmoc-ALFALFA-CONH2 (SEQ ID NO: 1)
was synthesized on a 0.1 mmol scale using 100 mg of resin. Based on
an initial estimate, a 2 minute DMF wash at 10 mL/min, a 2 minute
Fmoc deprotection step at 6 mL/min, and another DMF wash, and a 6
minute room temperature coupling with activated amino acid
delivered at 1 mL/min were chosen as the starting point for an
amino acid addition cycle. This sequence allowed efficient peptide
synthesis in 12 minutes per residue. The reverse phase (RP)-HPLC
trace for the crude peptide is shown in FIG. 2C.
[0091] After validating this approach, improved wash step, Fmoc
removal, and coupling times were determined. All subsequent studies
were carried out at 60.degree. C. to reduce the cycle time without
significantly increasing formation of side products. It is believed
that higher temperatures may improve synthetic outcomes and/or
reduce the total time required for each amino acid addition cycle.
The final synthetic timeline, which was used in all subsequent
experiments in this example, is shown in FIG. 3C. The final
synthetic timeline has a 2 minute DMF wash at 10 mL/min, a 20
second Fmoc deprotection step at 10 mL/min, another 2 minute DMF
wash, and a 30 second coupling step with activated amino acid
delivered at 12 mL/min. This approach was studied by synthesizing
the peptide ACP(65-74). This peptide served as a model to validate
the flow based SPPS platform, because ACP(65-74) was considered
difficult to prepare. It is believed that substantial reductions in
synthesis times could be achieved when synthesizing peptides that
are easier to prepare.
[0092] In conventional systems, the main synthetic impurity in the
synthesis of ACP(65-74) is a chromatographically resolved Val
deletion. The LCMS data for the synthesis of ACP(65-74) with the
flow based SPPS platform methodology, as well as two controls, is
shown in FIGS. 4A-4D. Using the above protocol and the HATU
coupling agent, a minor Val deletion product was observed. When
using HBTU, more Val deletion was observed, which is consistent
with prior reports. ACP(65-74) synthesized with the flow based SPPS
platform, but at room temperature, showed large Val and Gln
deletions, confirming that temperature is important. No major
differences between the product composition from the room
temperature synthesis and an analogous batch synthesis were
observed. Two additional "difficult" peptides, a conotoxin variant
and a fragment of the HIV-1 protease, were also synthesized. The
LCMS data is shown in FIGS. 5A-5B. Both of these peptides contained
cysteine residues that were observed to racemize during activation.
Therefore, model studies using the peptide GCF were carried out.
During the model study, several conditions that produced less than
1% diastereomer, as shown in FIGS. 6A-6E, were found. This level of
racemization is consistent with literature for Fmoc protocols.
[0093] Using the modified coupling conditions for cysteine, the
conotoxin variant and a fragment of the HIV-1 protease were
prepared on a 0.1 mmol scale. Eighty nine milligrams (53%) of the
crude conotoxin and 90 mg (43%) of the crude HIV-I protease
fragment were isolated. To explore the utility of the flow platform
in the preparation of synthetic proteins, a 58 residue tri-helical
protein based on the Z domain of protein A (referred to as the
affibody) was prepared. The synthetic strategy, which can be seen
in FIG. 7A, used peptide-hydrazides as thioester precursors for use
in native chemical ligation. Peptide hydrazides can be oxidized
with NaNO.sub.2 to form a C-terminal peptide-azide, which can react
with a thiol to form a peptide thioester. The LCMS data for the
crude synthetic peptides are shown in FIGS. 7B-D. Variants of these
peptides were also prepared using Boc in-situ neutralization
methods and the peptides were found to be of similar crude quality
(FIGS. 8A-8F). Retention time shifts are due to different
chromatographic conditions and slight variations in peptides
prepared for native chemical ligation with Boc and Fmoc strategies.
Each peptide for the affibody was purified (FIG. 9), the affibody
was then synthesized, and the highly pure, full-length affibody was
isolated after purification (FIG. 7E).
[0094] Although it was possible to implement this protocol in a
batch mode, the flow based platform overcame a number of
significant obstacles. First, the completely sealed reactor and a
preheat loop were immersed in a temperature controlled bath which
allowed reagents to be heated in a consistent and controlled manner
immediately before reaching the resin bed. This would be difficult
in some batch systems. Second, the use of a low volume reactor
(about 2.5 mL) and narrow tubing for delivery of solvents and
reagents allowed efficient washing with only 20 mL of solvent. In
contrast, batch mode automated and manual syntheses typically use
large volumes of solvent (about 70 mL per wash). Third, the flow
platform was assembled from common laboratory equipment at low cost
without machine or glass shop support. Fourth, high quality
peptides were obtained quickly without double coupling, double
deprotection, colorimetric tests, or resin mixing. During the
studies with ACP(65-74), no decrease in the Val deletion peptide
was observed after double coupling Val and double deprotecting the
preceding Gln. These additional steps are often employed in batch
mode syntheses. Finally, the flow based SPPS system was capable of
being adapted to larger synthetic scales by increasing the diameter
of the reactor. For example, the reactor diameter was doubled and
the resulting reactor used to synthesize ACP(65-74) on a 0.2 mmol
scale using exactly the same protocol. Another option for
increasing the synthetic scale was to simply increase the reactor
length. However, this strategy significantly increased the
backpressure, which may pose difficulties during synthesis. The
flow based SPPS platform in this example allowed for the rapid Fmoc
synthesis of polypeptides. It was found that, under flow at
60.degree. C., amide-bond formation and Fmoc removal were fast
(within seconds) and did not improve with increased reaction time.
Using the flow based Fmoc system in this example, three affibody
segments were able to be synthesized and cleaved in one working
day. By contrast, the production of similar peptides using
optimized Boc in-situ neutralization methods, with 15 minute cycle
times, required more than three days. In addition, the purified
peptides were ligated to generate synthetic proteins. This approach
allowed for the rapid production of highly pure, moderately sized
peptides that were easily ligated to obtain larger fragments.
Example 2
[0095] This example describes the determination of the deprotection
step time. Real-time monitoring of the effluent with an inline
UV-Vis detector allowed the deprotection step to be reduced in
length. The rate of Fmoc removal was investigated by monitoring the
UV absorbance of the reactor effluent at 304 nm. To determine the
minimum treatment time for robust Fmoc removal, the deprotection
solution was flowed in at 10 mL/min for 60 seconds, 30 second, 15
seconds, or 6 seconds. Twenty seconds at 10 mL/min was found to be
sufficient for complete Fmoc removal. Effective Fmoc removal was
also achieved during the 6 second steps.
[0096] In developing a N.alpha. deprotection protocol, piperidine
in DMF was selected as the standard deblocking reagent. A
concentration of 50% (v/v) in DMF was selected over the more common
20% (v/v) in DMF because the deprotection solution was diluted as
it entered the column. A higher concentration was therefore
desirable. The flow rate was set at 10 mL/min (maximum) to reach an
effective concentration in the minimum time. To determine the
length of the deprotection step, ALF peptide was synthesized with a
double deprotection of every residue, and the UV absorbance of the
effluent was monitored at 304 nm. Piperidine and DMF did not absorb
well at this wavelength, but piperidine-DBF, the deprotection
product, did. Therefore, the presence of a second peak after the
second deprotection indicated that the initial deprotection was
inadequate. No second peak was observed after 60 seconds, 30
seconds, and 15 seconds of deprotection, and only a very small peak
was observed after a 6 second initial deprotection. In all cases,
the first deprotection was at 10 mL/min, and the second was for one
minute at 10 mL/min. Since Fmoc removal has been reported to be
sequence dependent, a final deprotection time of 20 seconds was
selected. However, it is believed that the 6 second deprotection
step (and even faster deprotection steps) would be suitable for
many peptide synthesis processes. Additionally, it is believed
that, by increasing the flow rate of the deprotection agent and/or
the temperature of the stream comprising the deprotection agent,
robust Fmoc removal can be achieved in one second or less.
[0097] The double deprotection protocol had to be used to determine
deprotection time because it took significantly longer to wash the
piperidine-DBF adduct out of the resin than to remove the Na Fmoc
group. If the effluent was simply monitored until the absorbance
returned to near-baseline, most of the "deprotection" time would
have been spent washing the resin with deprotection reagent after
the deprotection was complete.
[0098] FIG. 10 shows the UV record of the incorporation of the
final eight residues during the conotoxin synthesis. Negative marks
represent manual actions. The scanned trace has been color enhanced
and a time-line added, taking zero to be the beginning of the
trace. The marks of one cycle have been annotated with 1 indicating
the end of the previous wash, 2 indicating the beginning of
coupling, 3 indicating the end of coupling, 4 indicating the start
of the first wash, 5 indicating the end of the first wash and start
of the deprotection, and 6 indicating the end of the deprotection
and start of the second wash. The quick connect was moved between 1
and 2, and between 3 and 4. Inconsistencies in cycle time and
missing marks were due to human error.
Example 3
[0099] This example describes the determination of the wash step
time. Real-time monitoring of the effluent with an inline UV-Vis
detector allowed the wash step to be reduced in time. The
efficiency of the wash step was systematically investigated by
monitoring the UV absorbance of the reactor effluent at 304 nm. The
time required to wash the amino acid out of the reactor as a
function of flow-rate was then investigated. It was determined that
the wash efficiency was principally determined by the total volume
of solvent used, with about 16 mL of DMF required to remove 99% of
the amino acid precursor. However, at flow rates greater than about
6 mL/min, marginally less solvent was required. It was concluded
that a 2 minute DMF wash at 10 mL/min was sufficient. Double
incorporation of amino acids, which could theoretically occur if
the DMF wash did not completely remove the amino acid or
deprotection solution, was not observed for the 2 minute wash time.
Increasing the wash volume did not improve the crude peptide
quality. It is believed that even faster wash times could be
observed by, for example, increasing flow rate, changing the
geometry of the inlet to reduce recirculation, and/or reducing the
aspect ratio of the reactor. Without wishing to be bound by theory,
it is further believed that, in some cases, there may not be an
unacceptable decrease in crude peptide quality if the wash is
reduced or eliminated. In some such cases, an amino acid addition
cycle may be employed that does not remove substantially all of an
activated amino acid prior to the deprotection step and/or does not
remove substantially all of the deprotection reagent prior to the
amino acid coupling step.
[0100] Visual observation of the reactor during wash cycles showed
recirculation and mixing of DMF wash solvent and coupling solution.
Other solvent exchanges showed the same behavior. Differences in
color and refractive indices allowed the direct observation of all
exchanges. Based on these observations, it was expected that the
wash efficiency was primarily dependent on the volume of solvent
used, as predicted by a continuous dilution model. To test this
theory, the UV absorbance of the reactor effluent was monitored at
304 nm during the triplicate synthesis of ACP(65-74). During each
synthesis, two consecutive residues were washed at 10 mL/min, two
at 7 m/min, two at 4 mL/min, two at 2 mL/min, and the final two at
1 mL/min. Wash rates were randomly assigned to blocks of amino
acids, ensuring that no block was washed at the same rate twice.
The time required for the detector to desaturate was measured for
each residue. Desaturation represents approximately 99% reduction
in amino acid concentration. This wash efficiency was selected
because the wash was essentially complete, but air and particulate
contamination in the detector were less significant than at lower
signal levels. The data are shown in FIG. 11. The exponent
(parameter b) was significantly below negative one. The value of
the exponent meant that less solvent was required to desaturate the
detector at higher flow rates. The relationship between
desaturation and flow rate was not consistent with the proposed
continuous dilution model.
[0101] Based on these results, a maximum flow rate (10 mL/min) was
selected for the wash. The wash time was set at two minutes, which
reliably reduced the final concentration of coupling solution to
0.2% of the initial concentration. This trend is valid with wash
rates up to at least 100 ml/min resulting in effective washing
observed in 10 seconds. It is believed that even faster washing
times could be achieved, for example, by increasing the wash liquid
flow rate. No double incorporation, a possible outcome of an
inadequate wash, was observed. The apparatus used did not provide a
direct way to monitor the removal of piperidine (the UV absorbance
is similar to DMF at accessible wavelengths), so the same wash
cycle was used for the second wash. If it was assumed that
piperidine is removed at the same rate as piperidine-DVB, the two
minute wash was an overestimate of the necessary wash time as shown
by the UV trace in FIG. 10. The total time of an amino acid
addition cycle may be reduced by substantially reducing the washing
step.
Example 4
[0102] This example describes the determination of minimum coupling
time. The effect of coupling time was investigated by synthesizing
two model peptides: LYRAG-CONH2 (SEQ ID NO: 2) and Fmoc-ALF-CONH2.
For each of five amino acid addition cycles, every amino acid was
coupled for a nominal time of 90 seconds, 45 seconds, 30 seconds,
15 seconds, or 7 seconds at 60.degree. C. as shown in FIG. 3. For
LYRAG-CONH2 (SEQ ID NO: 2), a significant increase in the Arg
deletion peptide was observed when all residues were coupled for 7
seconds. For Fmoc-ALF-CONH2, no significant difference in the
quality of the crude product as a function of coupling time was
found. Based on these results, a 30 second coupling time was
concluded to be sufficient.
[0103] It is generally known from the literature that, at room
temperature, amide-bond formation is 99% complete in less than 100
seconds using HBTU as a coupling agent. If it was assumed that the
reaction rate for this process doubled for every 10.degree. C.
increase in temperature, at 60.degree. C. amide bond formation
would be completed in about 6 seconds, which would have
significantly decreased amino acid addition cycle time. Thus, all
subsequent coupling studies were carried out at 60.degree. C. to
minimize the cycle time without significantly increasing formation
of side products. An important feature of this platform was the
ability to simply place the reactor and a preheat loop in a
temperature controlled water bath. The preheat loop allowed
reagents to be stored at room temperature and then immediately
heated before entering the reactor, which allowed the thermal
degradation of reagents to be minimized.
[0104] LYRAG (SEQ ID NO: 2) was selected as a model peptide to
determine the minimum coupling time, because the arginine deletion
could be monitored. For 90 seconds, 45 seconds, and 30 seconds
nominal coupling times, the coupling solution was delivered at 4,
8, and 12 mL/min, respectively. This flow rate allowed the delivery
of 2 mmol of amino acid. Flow rates above 12 mL/min were not
reliably obtainable in this system (although other systems could be
designed to include higher flow rates), so for the 15 second trial,
half of the coupling solution was used (1 mmol amino acid in 2.5 mL
0.4M HBTU in DMF, with 0.5 mL N,N-Diisopropylethylamine (DIEA)). At
a coupling time of 7 seconds, the time spent manually moving the
quick connect (5-6 seconds) was very significant, so 1.2 mL of
coupling solution was delivered. This volume was the volume of the
preheat loop, so the coupling solution did not reach the reactor
until it was cleared from the lines by the DMF wash. The wash, at
10 mL/min, took 7.2 seconds to clear 1.2 mL, giving a 7 second
coupling time. In the other runs, the 5 seconds to move the quick
connect was added to the nominal coupling time, as was the time
required to deliver about a 10% increase over the nominal volume of
coupling solution. The difference in the time taken for the DMF
wash solvent and coupling solution to clear the inlet line was
subtracted. More accurate coupling times were 93 seconds, 53
seconds, 39 seconds, 23 seconds, and 7 seconds. This does not
include the time required to wash the coupling solution from the
reactor. The seven second coupling showed increased Arginine
deletion, so the 30 second protocol was selected as a conservative
estimate. Fmoc-ALF was produced with the same procedure, and showed
no change in peptide quality with reduction in coupling time. Data
are presented in FIG. 3.
[0105] It is believed that using an automated system, using higher
flow rates, and using a higher temperature would substantially
reduce the coupling time.
Example 5
[0106] This example describes the minization of cysteine
racemization. The peptides Pn1A (A10L) conotoxin, HIV-1 PR(81-99),
and GCF were used to explore techniques to minimize cysteine
racemization.
[0107] In the initial syntheses of Pn1A (A10L) conotoxin and HIV-1
PR(81-99), cysteine was activated like all other amino acids (1 eq
HBTU, 2.9 eq DIEA), and significant diastereotopic impurities were
observed in the products. These were determined to be the result of
cysteine racemization. To investigate conditions that reduce
racemization, a model system, GCF, was selected because the
diasteromer formed upon racemization was resolved by RP-HPLC. The
standard synthetic procedure was used, except coupling time was
increased to one minute for 60.degree. C. runs and 6 minutes for
room temperature (RT) runs. Rink, Gly, and Phe were all activated
according to the standard procedure, with one equivalent of HBTU (5
mL, 0.4M) and 2.9 equivalents of DIEA (1 mL). For cysteine, various
activation procedures were used as summarized below. For procedures
that used less than 1 mL of DIEA, DMF was used to replace this
volume. In addition to the activation methods below, an authentic
diasteromer was produced using Fmoc-D-Cys(Trt)-OH and the
activation procedure of 5. TIC traces are shown for 4, 5, 6, 7, and
the authentic diastereomer in FIG. 6. Runs not shown were visually
indistinguishable from 4. In all cases, the activator, additive,
and 2 mmol amino acid were dissolved in 5 mL DMF, and additional
DMF was added as needed. The base was added immediately before use.
Reaction 8 employed an isolated C-terminal pentafluorophenyl (Pfp)
ester (Fmoc-Cys(Trt)-OPfp) without additional activators,
additives, or base. Table 1 summarizes the results, with
racemization quantified by integration of the extracted ion
current. This enables quantification of racemization below the TIC
baseline. The results obtained were consistent with previous
reports.
TABLE-US-00001 TABLE 1 Reaction conditions and racemization
results. Reaction Activator Additive Base Temp Racemization 1 HBTU
None DIEA 60.degree. C. 10% (1 eq) (2.9 eq) 2 HBTU HOBt DIEA
60.degree. C. 18% (1 eq) (1 eq) (2.9 eq) 3 HBTU None DIEA Cys room
temp 11% (1 eq) (2.9 eq) G and F 60.degree. C. 4 HBTU None DIEA
Room 10% (1 eq) (2.9 eq) temperature 5 HBTU None DIEA 60.degree. C.
1% (1 eq) (0.9 eq) 6 HBTU HOBt DIEA 60.degree. C. 1% (1 eq) (1 eq)
(0.9 eq) 7 DCC HOBt None 60.degree. C. 1% (0.9 eq) (1.1 eq) 8 OPfp
None None 60.degree. C. 1%
[0108] FIGS. 6A-6E shows GCF produced with various cysteine
activation schemes. The peak eluting between the desired product
and diastereomer in A and B was hydrolysis of the C-terminal
carboxamide. The diastereomer was barely visible. Conditions are
listed in table 1. The conditions in FIG. 6A-D show chromatograms
for reactions (a) 5, (b) 7, (c) 8, (d) 4, and the (e) the authentic
Gly-D-Cys-L-Phe. The total ion current is displayed in each
chromatogram.
Example 6
[0109] This example describes the synthesis of an affibody.
Throughout this section, ligation buffer refers to a 6M GnHCl, 0.2M
Sodium Phosphate buffer at the specified pH; buffer P is a 20 mM
Tris, 150 mM NaCl solution at pH=7.5.
[0110] Oxidation chemistry was used to ligate three fragments into
a synthetic, 58 residue protein. Thiazolidine was found to be
unstable in the conditions used, so 9.6 mg of fragment
Thz-[28-39]-CONHNH.sub.2 was converted to a free N-terminal
cysteine by treatment with 83 mg methoxyamine hydrochloride in 5 mL
of ligation buffer at pH=4 overnight. Quantitative conversion was
observed. The N-to-C assembly shown in FIG. 5A was employed instead
of the C-to-N synthesis used when thioesters are accessed directly.
Fragment [1-27]-CONHNH.sub.2 was oxidized to the C-terminal azide
by drop wise addition of 0.1 mL of 200 mM aqueous NaNO.sub.2 to a
solution of 11 mg purified fragment [1-27]CONHNH.sub.2 in 1 mL
ligation buffer at pH=3 and 0.degree. C. The reaction proceeded for
20 minutes at 0.degree. C., and was then quenched by the addition
of 172 mg 4-mercaptophenylacetic acid (MPAA) and 34 mg
tris(2-carboxyethyl)phosphine-HCl (TCEP.HCl) dissolved in 4.4 mL
ligation buffer (pH=7, room temperature (RT)). To the resulting
thioester, 3.4 mL of the crude methoxyamine treated fragment
Thz-[28-39]-CONHNH.sub.2 were added. After a two hour RT ligation,
one half of the crude reaction mixture was purified by RP-HPLC,
with 2 mg of highly pure material recovered. Of this, 0.6 mg were
oxidized by dissolution in 0.1 mL of ligation buffer and drop wise
addition of 0.01 mL of 200 mM aqueous NaNO.sub.2 at 0.degree. C.
The reaction proceeded for 26 minutes, and was then quenched by
addition of 3.1 mg MPAA and 0.78 mg TCEP.HCl dissolved in 0.1 mL
ligation buffer (pH=7, RT). The pH was adjusted to 7 and 0.3 mg of
fragment Cys-[40-58]-CONH.sub.2 were added to the reaction mixture.
After a two hour RT ligation, the mixture was diluted with 0.21 mL
buffer P, then a further 0.63 mL buffer P to fold the resulting
affibody. The crude mixture was concentrated over a 3 kDa membrane
to a final volume of 0.075 mL. The crude, folded protein was
diluted with 36 mg TCEP.HCl in 3 mL buffer P and purified to
homogeneity (FIG. 9).
[0111] FIGS. 9A-9E show LC-MS chromatograms of the purified
affibody synthesis intermediates and final product. FIGS. 9A-9E are
chromatograms of (a) fragment [1-27]CONHNH.sub.2, (b) fragment
Thz-[28-39]-CONHNH.sub.2, (c) fragment Cys-[40-58]-CONH.sub.2, (d)
ligation fragment [1-39]-CONHNH.sub.2, and (e) the final
affibody.
Comparative Example 6
[0112] This example describes the synthesis of an affibody using a
conventional manual Boc in-situ neutralization method.
[0113] Comparable affibody fragments were synthesized using manual
Boc in-situ neutralization methods. LC-MS data for these crude
peptides is shown in FIGS. 8A-8F, next to the crude peptides
synthesized on the flow based SPPS platform. In all cases, the
quality was comparable. Retention time shifts are due to a change
in chromatographic conditions and slightly different peptides
prepared for ligation with Boc and Fmoc strategies.
Example 7
[0114] This example describes the design of the system used for
flow based SPPS. Throughout this example the system is referred to
as the synthesizer. A schematic of the synthesizer is shown in FIG.
2A. An HPLC pump was used to deliver either methanol purge solvent
for washing the pump heads, reactor, and UV detector after use; DMF
wash solvent for removal of reagents and byproducts during
synthesis; or 50% (v/v) piperidine in DMF for deprotection of the
N-terminus. The positions of valves 1 and 2 determined which fluid
was delivered. A syringe pump was used to deliver coupling
solution. To switch between the syringe pump and HPLC pump, a quick
connect was manually moved from the outlet of the HPLC pump to the
syringe on the syringe pump. A valve would have been generally
ineffective because the line between the syringe pump and valve
would retain coupling solution, causing incorrect incorporation in
the next cycle. The column and a 1.2 mL preheat loop (not shown)
were submerged in a water bath to maintain a constant 60.degree. C.
Valves 3 and 4 selected a high pressure bypass loop used to clear
the UV detector when it was clogged with precipitates, such as the
urea byproduct of DCC activation encountered during cysteine
racemization studies. The loop was also used to purge the detector
without the column in line.
[0115] A Varian Prostar 210 HPLC pump, KD Scientific KDS200 syringe
pump, Varian Prostar 320 UV detector set to 304 nm, Amersham
Pharmacia Biotech chart recorder, and VWR 39032-214 water bath were
used in the synthesizer. The HPLC pump delivered about 95% of the
nominal flow rate. Disposable 10 mL syringes (BD 309604) were used
to deliver coupling solutions. Valve 1 was a Swagelok 1/8'' 3-way
valve (S5-41GXS2). The other valves in the system were Swagelok
1/16'' 3-way valves (SS-41GXS1). The methanol, DMF, and 50% (v/v)
piperidine lines through valve 1 up to valve 2 were 1/8'' OD,
1/16'' ID FEP (Idex 1521). The line between valve 3 and 4 was
1/16'' OD, 0.010'' ID peek (Idex 1531). All other lines were 1/16''
OD, 0.030'' ID PFA (Idex 1514L). To attach the 1/8'' wash and
deprotect lines to the 1/16'' inlets of valve 2, Swagelok 1/8'' to
1/16'' (SS-200-6-1) reducing unions were used, followed by a short
section of 1/16'' tubing. All lengths were minimal, except the
tubing between the quick connect and the reactor. This included a
2.6 m (1.2 mL) coil which was submerged along with the reactor and
served as a preheating loop to ensure that reactants were at
60.degree. C. before reaching the reactor. Whenever tubing had to
be joined, Swagelok 1/16'' unions were used (SS-100-6). These were
used to attach the preheat loop, join the outlet of the column to a
line from valve 4, and repair a severed bypass loop. The manually
changed quick connect was a female luer to 10-32 female HPLC
fitting (Idex P-659). This connected directly to the syringes on
the syringe pump or to a mating male luer to 10-32 female fitting
on the line from valve 3 (Idex P-656). The connection between the
UV detector and chart recorder was a data link (three 18 ga
insulated copper wires).
[0116] FIG. 2B shows the reactor assembly. The reactor consisted of
a tube with standard compression fittings on each end (3/8'' to
1/16'' reducing unions). On the downstream end there was also a
frit. This was positioned by a support designed to fit inside the
reactor and seat against the bottom of the fitting on that end.
Various frit porosities were used. The part number below was for a
20 micron frit, the most commonly used. The body was a 3.5 inch
segment of PFA tubing with outer diameter 3/8'' and inner diameter
1/4''. The frit was a 1/4'' sintered stainless steel disk 1/16''
thick. The frit support was a 0.5'' length of 1/4''OD PTFE tubing.
As the fittings were tightened, the nut compressed the ferrule
against the fitting body, sealing the reactor body to the fitting
body. This also compressed the reactor body against the frit,
forming an internal seal against the frit. The reactor body and
frit were purchased from McMaster-Carr as part numbers S1805K73 and
94461314, respectively. The nut, ferrule, and fitting body are
available as a set with the 1/16'' nut and ferrule from Swagelok as
part number SS-600-6-1. Replacement ferrules are available as
SS-600-SET. The reactor was assembled by first cutting the body and
frit support to length, ensuring the ends were square. A sharp
razor blade and steady hand were used for these operations. Next,
the outlet (downstream) end was assembled. The frit was placed on a
solid, clean surface and the reactor body was pressed onto it.
After verifying that the frit was square and flush with the end of
the reactor, the frit support was pushed in slightly, pushing the
frit up towards its final position. Firmly seating the reactor body
in the fitting body forced the frit to its final position. It was
verified that the frit was square and properly positioned under the
ferrule, then the fitting was installed according to the
manufacturer's instructions. Once sealed, the frit could not be
removed and reseated. Finally, the inlet fitting was installed
according to the manufacturer's instructions. A high pressure
reactor with a stainless steel body was also built. In this case,
the downstream fitting had to be tightened well beyond
specification to affect a seal with the frit. The reactor was
typically replaced every 3-8 syntheses. When replacing the reactor,
the ferrules, frit and reactor body were not reused. All other
parts were reused. The nuts were recovered by cutting the reactor
body in half.
[0117] To load the reactor, the upstream fitting body was removed
and a slurry of resin in methanol was pipetted in. The reactor was
completely filled with methanol, and the fitting body was
reinstalled. The inlet line and preheat loop were filled with
solvent by attaching them to the quick connect and running the HPLC
pump before attaching them to the reactor. The reactor was then
kept upright in the water bath so that any small bubble would move
to the top and not interfere with wetting the resin. Before the
first coupling, the resin was washed for two minutes with DMF at 10
ml/min.
Example 8
[0118] This example describes the design of a large-scale reactor,
used for flow based SPPS in Examples 10-11 and 13-17 that allowed
faster cycle times and increased synthetic scale. Throughout the
examples, this reactor is referred to as the "second generation"
reactor, in contrast to the "first generation" reactor described in
Example 7.
[0119] FIG. 12 shows the larger reactor. The design principles of
the small-scale reactor (i.e. the first generation reactor) used
for all syntheses in Examples 1-7 translated directly to larger
scales. In order to preserve comparable cycles, however, the volume
of the reactor had to be constant. Two problems were encountered
when scaling up to a 5/8'' OD, 1/2'' ID tube. First, there were no
standard 5/8'' to 1/16'' compression fittings. Second, the minimum
distance between 5/8'' fittings is quite large, meaning there is a
large minimum volume. To overcome the first problem, a 5/8'' to
3/8'' fitting followed by a 3/8'' to 1/16'' fitting was used, but
this necessitated a joining length of 3/8'' tubing that greatly
increased the already large volume of the reactor.
[0120] To reduce the reactor volume, a 316SS insert was machined
that consisted of a nominal 1/2'' OD segment followed by a 3/8'' OD
segment with a 1/4'' through hole. A 5/8'' to 3/8'' reducing union
was bored out to give a 3/8'' through hole, the insert was seated,
and the 3/8'' ferrule swaged on. After this, the insert could not
be separated from the fitting. When installed, the 1/2'' part of
this insert-fitting sat in the top of the reactor and limited the
volume.
[0121] This was effective, but there was still a large volume from
the 1/4'' through hole. This volume was reduced by inserting a
1/4'' OD, 1/8'' ID PFA tube and cutting it flush. To further reduce
the volume, a 1/8'' OD, 1/16'' ID PFA tube was inserted by heating
and drawing a section of tubing to a narrower diameter, threading
it through, and pulling until all tubing in the insert was of the
proper diameter. Both sides of the tube were cut flush and the
drawn section was discarded. A 3/8'' to 1/16'' reducing union was
installed on the open end of the 3/8'' segment to interface with
the rest of the system. This insert-fitting is pictured in FIG. 12A
(left). To prevent the upstream insert fitting from becoming
permanently sealed into the tube like the frit, the nominal 1/2''
segment was machined to 0.496'' and polished.
[0122] A similar piece was machined for the outlet side, with a
1/2'' section the proper length to seat the frit under the ferrule.
To prevent all of the solvent from being forced through a small
central section of the frit, a 3/8'' diameter step 0.05'' deep was
cut. The bottom of this step tapered to a 1/8'' through hole at 31
degrees from horizontal (a standard drill bit taper). A 1/8'' OD,
1/16'' ID PFA tube was inserted to further limit the volume. The
1/2'' section of the outlet insert positioned the frit and sat
largely below the ferrule, so a standard finish was adequate. The
one pictured in FIG. 12A was PTFE, and was installed in a bored
through 5/8'' to 3/8'' reducing union in exactly the same way as
the upstream insert. Subsequent reactors, including the one in the
cutaway pictured in FIG. 12F, used stainless steel outlet inserts.
A 3/8'' to 1/16'' reducing union was installed on the open end of
the 3/8'' segment to interface with the rest of the system.
[0123] Tubing was used to limit the internal volume of the inserts,
rather than directly making inserts with small holes, to simplify
fabrication.
[0124] To assemble the reactor, the frit was pressed in and the
downstream insert-fitting installed as a regular fitting. The
upstream insert-fitting was then installed as a regular fitting.
Despite cutting it undersize and polishing, the upstream
inset-fitting was very tight and difficult to remove. For
subsequent reactors, an aluminum spacer, shown in FIG. 12B, was
used to allow the reactor to have a consistent volume. The spacer
set the internal volume to 2 mL and enabled reproducible assembly
of the reactor. Furthermore, the spacer helped remove the inlet
insert-fitting after synthesis. The spacer prevented the nut from
moving down, and instead ejected the insert-fitting when the nut
was turned. A vertical window was added to the spacer to maintain
adequate optical access. A picture of the assembled large reactor
is shown in FIG. 12D.
[0125] To load the reactor, the inlet insert-fitting was removed,
the resin was added dry, and the reactor filled with methanol. The
inlet insert-fitting was then attached to the heat exchanger and
purged with methanol. Without removing it from the heat exchanger,
the purged inlet insert-fitting was installed, causing excess
methanol to exit the reactor through the outlet insert-fitting. If
there was a large bubble, the reactor was turned upside down (with
the inlet below the outlet) and purged. If this failed to dislodge
the bubble, the reactor was disassembled and the loading procedure
was repeated. To remove the resin after synthesis, a syringe filled
with 10 mL of air was attached to the luer-lock quick connect on
the heat exchanger inlet (where reagent syringes are attached) and
used to deliver the air. This removed solvent from the heat
exchanger, reactor, and waste line. The heat exchanger and waste
line were then disconnected from the reactor and the inlet
insert-fitting removed. The resin was suspended in DCM and decanted
into a fritted syringe (Toviq), washed four times with DCM, and
either cleaved immediately or dried under reduced pressure for
storage.
Example 9
[0126] This example describes techniques used to reduce pressure in
the first generation reactor. Pressure drop was inherently caused
by the resin. Pressure drop was overcome by employing a rest period
after high pressure flows or using a large reactor.
[0127] A low pressure polymer reactor was used, so an overpressure
alarm on the HPLC pump was set to shut off the pump at 240 psi,
which was occasionally triggered. When the alarm was triggered, the
system was allowed to rest for 30 seconds, and the pumps were
restarted without further incident. During this resting phase, the
resin visibly expanded. By observing the HPLC pump pressure, it was
concluded that if too much pressure was applied to the beads, they
begin to compact. This increased the pressure drop across the bed,
and the rate of compaction, which quickly triggers the over
pressure alarm. Similar 1% divinyl benzene crosslinked polystyrene
resin available for gel permeation chromatography from Bio-Rad was
recommended for gravity driven separations only, because it is very
soft once swollen.
[0128] When the reactor was disassembled immediately after such an
event, the resin looked like a solid block and, when probed with a
pipette tip, felt like a hard mass. It was difficult to immediately
pipette it out. After a few tens of seconds, the resin relaxed and
could be pipetted out. A high pressure stainless steel reactor was
built and tested, but the very high pressure necessary to maintain
a high flow through a compacted bed (>1000 psi) was reminiscent
of previous continuous flow SPPS that struggled with extrusion of
the resin through the frit.
[0129] It was believed that the initial compaction took place at
the boundary of the frit and the resin, such that the resin was
able to mechanically block the pores of a course frit with
relatively little deformation. To test this theory, the original 40
micron frit was replaced with a 20 micron frit, and, in more
limited trials, 10 micron frits and 2 micron frits. Smaller pores
did not eliminate the problem, but seemed to qualitatively reduce
its severity. From this it was concluded that the problem was
inherent in the resin, and can only be eliminated by running at
lower flow rates or reducing the bed height (using smaller scales
and/or larger reactors).
[0130] The use of harder, more highly crosslinked resin has been
reported, but the resulting peptides were of inferior quality. The
solution used here was to wait 30 seconds following a high pressure
event. This was effective and expedient, allowing progress on a
reasonable scale without further optimizing the dimensions of the
reactor. Trials with a 1/2'' ID reactor (described in Example 8)
showed no overpressure with up to 200 mg of resin, operating on the
same cycle.
[0131] To overcome these problems, accelerate synthesis, and
increase synthetic scale, the large scale reactor described in
example 8 was constructed.
Example 10
[0132] This example describes the preparation of
ALFALFA-CONHNH.sub.2 (SEQ ID NO: 48) in six minutes. A high
capacity pump head for the HPLC pump used in previous examples was
used to deliver 100 ml/min of DMF during the wash step, 100 ml/min
of 50% piperidine in DMF during the deprotection step, and 12
ml/min of activated amino acids during the coupling step. The
reactor and preheat loop were maintained at 60.degree. C. by
immersion in a water bath.
[0133] The apparatus shown in FIG. 13 was constructed. Reservoir 1
contained activated alanine, reservoir 2 contained activated
leucine, reservoir three contained activated phenylalanine,
reservoir 4 contained 50% piperidine in DMF, and reservoir 5
contained DMF. Each activated amino acid was prepared by combining
50 ml of 0.4M HBTU in DMF with 20 mmol Fmoc protected amino acid.
Immediately before the start of the run, 10 mL of DIEA was added to
each of the amino acid reservoirs. To obtain the desired flow
rates, 1.5 bars of nitrogen head pressure was applied to each
reservoir. All tubing upstream of the pump was 1/8'' OD, 1/16'' ID
PFA. The three way valves were Swagelok 1/8'' three way valves. The
common lines of three way valves were routed into a switching valve
(Valco C25-6180) which selected between the reagents. All valves
were manually controlled. The pump was a Varian Prostar 210 with a
100 ml/min pump head. The preheat loop was 1.8 m of 1/16'' OD,
0.030'' ID PFA tubing. The reactor used was the larger reactor
shown in FIG. 12 and described in Example 8. The reactor contained
120 mg of chlorotrityl hydrazide functionalized polystyrene resin,
prepared from commercial chlorotrityl chloride resin using standard
methods known to those of ordinary skill in the art. Using the
larger reactor helped in maintaining a manageable pressure drop at
100 ml/min.
[0134] One synthetic cycle was performed as follows. First a 20
second coupling was performed at 12 ml/min. The multiport valve was
set to the desired amino acid and the three way valve was set to
amino acid. All other three way valves were set to DMF. After
twenty seconds, the selected three way valve was switched from
amino acid to DMF and the pump flow rate was set to 100 ml/min.
After five seconds, the multiport valve was switched to piperidine.
After another five seconds, the selected three way valve was
switched from DMF to piperidine. After 10 seconds the selected
three way valve was switched back to DMF. After five seconds, the
multiport valve was moved to the next desired amino acid. After
another five seconds, the flow rate was reduced to 12 ml/min and
the selected three way valve was switched from DMF to the next
desired amino acid, starting the next cycle. The total time for
each step was as follows: 20 second coupling, 10 second wash, 10
second deprotection, and 10 second wash. The total time for each
cycle was 50 seconds. The total ion chromatogram from the LC-MS
analysis of the crude material is shown in FIG. 14.
[0135] All of the above-listed times are believed to be
conservative estimates of what would be required to achieve 99%+
yields. It is now known that at a flow rate of 20 ml/min the
deprotection is finished in 5 seconds, and it is expected that at
100 ml/min the deprotection requires substantially less than 5
seconds. Longer peptides, such as the common model peptide
ACP(65-74), could be prepared, for example, by integrating
additional 3-way valves. The general strategy described in this
example is expected to be viable for the production of any peptide,
including those produced using the cycles described in Example
1.
Example 11
[0136] This example describes an improved synthesis scheme, in
which the synthesis times (relative to Example 1) were
substantially reduced. The cycle time for the synthesis in this
example was less than 3 minutes. To reduce the cycle time relative
to Example 1, the wash step was adjusted. All tubing upstream of
the pump was replaced with 1/8'' OD 1/16'' ID PFA, and the two
valves upstream of the pump were replaced with 1/8'' Swagelok three
way valves. All tubing lengths except the preheat loop were
minimal, and the reactor described in Example 8 was used. All other
system components were substantially unchanged relative to Example
1. Unless explicitly mentioned below, all procedures remained the
same, relative to Example 1.
[0137] The larger tubing and a high capacity pump head (maximum 50
ml/min) were used to deliver DMF and deprotection reagent at 20
ml/min. As expected based on FIG. 11, a one minute wash at 20
ml/min proved to be adequate in all cases. Furthermore, a 5 second
deprotection step was found to be adequate at these flow rates. The
coupling step was unchanged. This yielded a total cycle time of 2
minutes 35 seconds to about 2 minutes and 50 seconds, depending on
the speed with which the manual steps are performed. The wash was
set at 20 ml/min instead of the maximal 50 ml/min because most
users have difficulty manually operating the system at the higher
flow rate. It is expected that automation can be used to overcome
this human limitation and allow for the implementation of cycles of
about 10 seconds per residue with sufficiently large pumps.
[0138] Two chromatograms of peptides made using this cycle are
shown in FIG. 15. In each case the main peak is the desired
product. These are typical results for peptides of this length.
Example 12
[0139] This example describes in more detail the materials used in
Examples 1-11 and 13-18 and the methods used in Examples 1-11.
[0140] 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HBTU),
2-(7-Aza-1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HATU), hydroxybenzotriazole (HOBT), and
Na-Fmoc protected amino acids were from Chem-Impex International,
IL, NovaBioChem, Darmstadt, Germany, and Peptide Institute, Japan.
4-methylbenzhydrylamine functionalized crosslinked polystyrene
(MBHA resin) and p-Benzyloxybenzyl alcohol functionalized
crosslinked polystyrene (Wang resin) were from Anaspec, CA.
N,N-Dimethylformamide (DMF), dichloromethane (DCM), diethyl ether,
methanol (MeOH) and HPLC-grade acetonitrile were from VWR, PA.
Triisopropyl silane (TIPS) and 1,2 Ethanedithiol were from Alfa
Aeser, MA. Trifluoroacetic acid (TFA) was purchased from NuGenTec,
CA, Halocarbon, NJ, and Sigma-Aldrich, MO. Solvents for LC-MS were
purchased from TJ Baker and Fluka. All other reagents were
purchased from Sigma-Aldrich, MO.
[0141] Common solvent mixtures used throughout these experiments
were: 0.1% (v/v) TFA in water (A), 0.1% (v/v) Formic acid in water
(A'), 0.1% (v/v) TFA in acetonitrile (B), and 0.1% (v/v) formic
acid in acetonitrile (B').
[0142] All peptides except the ACP(65-74) batch in FIG. 4D were
synthesized on the flow based SPPS system. All peptides except
ACP(65-74) batch and ACP(65-74) flow RT in FIGS. 4D and 4C were
synthesized at 60.degree. C., with reagents preheated immediately
before use via a preheat loop (see synthesizer design). One
synthetic cycle consisted of an amino acid exposure step (e.g.,
amide bond formation, also referred to as coupling in the
examples), an amino acid removal step (e.g., removal of the
coupling reagent, also referred to as a wash step in the examples),
a deprotection agent exposure step (e.g., Na Fmoc removal, also
referred to as deprotection in the examples), and a deprotection
agent removal step (e.g., removal of deprotection reagent and
reaction product, piperidine-dibenzofulvene (piperidine-DBF), also
referred to as a wash in the examples).
[0143] Unless noted, coupling was performed by delivering the
following coupling solution at 12 ml/min (for approximately 30
seconds) in Examples 1-7. The activated coupling solution consisted
of 2 mmol of Na-Fmoc and side chain protected amino acid dissolved
in 5 ml of 0.4M HBTU in DMF and 1 mL of DIEA. Cysteine was
dissolved in 5 mL 0.4M HBTU in DMF, 0.687 mL neat DMF, and 0.313 mL
DIEA. In both cases, amino acids were dissolved in HBTU solution up
to several hours before use, and DIEA was added within two minutes
of use. Volumetric measurements were made at RT (20.degree. C.).
The ACP(65-74) shown in FIG. 4A was synthesized by substituting
HATU for HBTU in the above solution.
[0144] Next, the coupling solution was removed with 20 mL of DMF
delivered at 10 mL/min over 2 minutes, and then the N.alpha.-Fmoc
protection group was removed with 3.3 mL of 50% (v/v) piperidine in
DMF delivered at 10 mL/min over 20 seconds. Excess piperidine and
piperidine-DBF were removed with 20 mL of DMF delivered at 10
ml/min over 2 minutes to complete one cycle.
[0145] All peptides were synthesized on 100 mg of 1% divinyl
benzene crosslinked polystyrene resin. To produce C-terminal
carboxamide peptides, MBHA functionalized resin with a loading of 1
mmol per gram was used, and the first residue coupled was the TFA
labile Rink linker. To produce C-terminal hydrazide peptides for
ligation, Wang resin, functionalized as below, was used. The
loading was 0.6 mmol/g (0.06 mmol scale).
[0146] Non-cysteine containing carboxamide peptides were cleaved
from the resin and side-chain deprotected by treatment with 2.5%
(v/v) water and 2.5% (v/v) TIPS in TFA for two hours. Cysteine
containing carboxamide peptides were cleaved from the resin and
side chain deprotected with 2.5% (v/v) EDT, 2.5% (v/v) TIPS, and 1%
(v/v) water in TFA for two hours. Hydrazide peptides were cleaved
with 5% (v/v) EDT, 5% (v/v) TIPS, and 2.5% (v/v) water in TFA for
two hours. In all cases, the resin was removed and compressed air
was used to evaporate the cleavage solution to dryness at RT. The
resulting solids were washed three times with cold diethyl ether,
dissolved in 50% A/50% B (v/v), and lyophilized. Side chain
protection was as follows: Arg(Pbf), Tyr(tBu), Lys(Boc), Asp(OtBu),
Gln(Trt), Ser(tBu), His(Trt), Asn(Trt), Trp(Boc), Glu(OtBu),
Thr(tBu), Cys(Trt).
[0147] The Wang resin was functionalized as follows: 5.47 g Wang
resin was added to a 500 ml round bottom flask and suspended in 98
mL of DCM and 1.12 mL of N-Methyl morpholine. This was stirred in
an ice bath for 5 min and 2.03 g p-nitrophenol chloroformate was
added as a powder. This mixture was stirred for 8.5 hours. The ice
in the bath was not replenished, which allowed the reaction to
slowly reach RT. The mixture was filtered and the solids washed
with DCM, DMF, MeOH, and DCM to give a white resin. The resulting
resin was placed in a clean 500 ml round bottom flask in an ice
bath, and suspended in a prepared mixture of 210 mL DMF, 54 mL DCM,
and 1.1 mL hydrazine monohydrate pre-chilled to 0.degree. C. This
yielded a bright yellow solution. The reaction proceeded for 18
hours in an ice bath that was allowed to melt. The mixture was then
filtered, and the solids washed as before to give
hydrazine-functionalized Wang resin.
[0148] All peptides were analyzed on an Agilent 6520 Accurate Mass
Q-TOF LC-MS under one of four conditions, as indicated below.
Condition 1: an Agilent C3 Zorbax SB column (2.1 mm.times.150 mm, 5
.mu.m packing) was used (condition 1). The flow rate was 0.4 mL/min
of the following gradient: A' with 1% B' for 3 minutes, 1-61% B'
ramping linearly over 15 min, and 61% B' for 4 minutes. Condition
2: For GCF and LYRAG (SEQ ID NO: 2) as well as other peptides, an
Agilent C18 Zorbax SB column (2.1 mm.times.250 mm, 5 .mu.m packing)
was used. The flow rate was 0.4 mL/min of the following gradient:
A' with 1% B' for 5 minutes, 1-61% B' ramping linearly over 15 min,
and 61% B' for 4 minutes. Condition3: an Agilent C3 Zorbax SB
Column (2.1 mm.times.150 mm, 5 .mu.m packing) was used with a flow
rate of 0.8 mL/min of the following gradient: A' with 5% B' for 3
minutes, 5-65% B' ramping linearly over 9 min, and 65% B' for 1
minute. Condition 4: An Agilent C3 Zorbax SB column (2.1.times.150
mm, 5 .mu.m packing) was used with a flow rate of 0.4 mL/min of the
following gradient: A' with 5% B' for 3 minutes, 5-95% B' ramping
linearly over 15 min, and 95% B' for 4 minutes. Unless noted,
peptides were analyzed under condition 1. LYRAG (SEQ ID NO: 2) used
in the coupling time study and GCF used in the cysteine activation
studies were analyzed under condition 2. Peptides in FIGS. 23C,
21A-B, 20, and 17 were analyzed under condition 3. Peptides in
FIGS. 23E, 22, and 21C were analyzed under condition 4. Total ion
current is displayed in all chromatograms.
[0149] The peptides were purified as follows. Crude peptides were
dissolved in 95% A/5% B (v/v) and purified on a Waters preparative
HPLC with an Agilent Zorbax SB C18 column (21.2 mm.times.250 mm, 7
.mu.m packing), a linear gradient from 5%-45% B in A over 80 min,
and a flow rate of 10 mL/min. The crude affibody Fragment 1-39
ligation product was purified on a Beckman System Gold
semi-preparative HPLC with a Zorbax C18 column (9.4 mm.times.250
mm, 5 .mu.m packing), a linear gradient from 10% to 55% B in A over
90 minutes, and a flow rate of 5 mL/min. The final affibody was
purified on the same system with the same gradient, using a Jupiter
C18 column (4.6 mm.times.250 mm, 5 .mu.m packing) and a flow rate
of 2.3 mL/min.
[0150] For all purifications, one minute fractions were collected
and screened for the correct mass on a PerSpective Biosystems
Voyager-DE MALDI-TOF using 2 .mu.L of the fraction co-crystallized
with 2 .mu.L of 50% A'/50% B' (v/v) saturated with
alpha-cyano-4-hydroxycinnamic acid matrix. The purity of pooled
fractions was confirmed by LC-MS, as above.
[0151] The UV detector response was quantified as follows. To
understand the UV traces produced and the wash efficiencies they
represent, the response of the UV detector was quantified. To
determine the approximate concentration of amino acid in the UV
traces, a serial dilution of Fmoc-Ala-OH coupling solution was
prepared. The initial concentration of amino acid was about 0.3M (2
mmol in 6.5 mL total volume) 10.times., 100.times., 1000.times.,
10,000 and 100,000.times. dilution standards were prepared and
injected directly into the UV detector. The 100.times. dilution
(3.times.10.sup.-3M) was just below saturation. The 10,000.times.
dilution (3.times.10.sup.-5M) standard was just above baseline,
about 1% of scale, as expected. The 100,000.times. dilution was
below the detection limit. The highly reproducible washout traces
(FIG. 10) show that this is representative of all amino acids
(qualitatively different traces between cycles would be expected if
the absorbance was vastly different).
Example 13
[0152] This example describes the general conditions for peptide
synthesis with the second generation reactor. This method was
sufficiently robust that all of these peptides were synthesized
without UV monitoring of the reactor effluent.
[0153] Increasing the diameter of the reactor reduced the
backpressure, and maintaining a comparable volume allowed the same
volumes of solvents and reagents to be used as the first generation
reactor. The second generation reactor accommodated up to 200 mg of
resin and accommodated flow rates up to 100 mL/min. More resin was
not used, because the selected resin swelled as the peptide was
elongated and the volume limiting inserts restricted the swollen
volume of the resin to 2 mL. Higher flow rates were not tested, but
the observed backpressure indicated that higher flow rates could be
achieved.
[0154] With the second generation reactor, cycle time was reduced
by washing at a higher flow rate. As expected, the required wash
time continued to decrease with increasing flow rate, with 99% of
the amino acid removed in 36 seconds at 20 mL/min and 20 seconds at
40 mL/min. However, to accommodate manual operation and allow
operators adequate time to prepare each subsequent amino acid, one
minute washes at 20 mL/min or 30 second washes at 40 mL/min were
used.
[0155] Peptides in FIGS. 16A-B, 17, 18 A-B, 19 A-C, 20, 21, 22A,
and 24A were synthesized using the second generation reactor
described in Example 8 and the synthesizer described in Example 1
at 60.degree. C. under conditions described in this Example, with
reagents heated immediately before use via a coil of tubing in a
water bath. ACP(65-74) shown in FIG. 16C was synthesized as below,
but at room temperature, and ACP(65-74) shown in FIG. 16D was
synthesized in a standard glass reactor. In all cases, one
synthetic cycle consisted of amide bond formation (coupling),
removal of coupling reagent (wash), Na Fmoc removal (deprotection),
and removal of the deprotection reagent and reaction product,
piperidine-dibenzofulvene (piperidine-DBF) (wash).
[0156] Unless noted, coupling was performed by delivering the
following coupling solution at 6 mL/min (for approximately 30
seconds). The coupling solution consisted of 1 mmol of Na-Fmoc and
side chain protected amino acid dissolved in 2.5 mL of 0.4 M HBTU
in DMF and 0.5 mL of DIEA. Cysteine was dissolved in 2.5 mL 0.4 M
HBTU in DMF and 0.157 mL DIEA. In both cases, amino acids were
dissolved in HBTU solution up to several hours before use, and DIEA
was added within two minutes of use. Volumetric measurements were
made at RT (18-20.degree. C.). The ACP(65-74) shown in FIG. 16A,
and the protease sites in FIG. 17 were synthesized by substituting
HATU for HBTU in the above solution. Next, the coupling solution
was removed with 20 mL of DMF delivered at 20 mL/min over 1 minute,
and then the Na-Fmoc protecting group was removed with 6.6 mL of
50% (v/v) piperidine in DMF delivered at 20 mL/min over 20 seconds.
Excess piperidine and piperidine-DBF were removed with 20 mL of DMF
delivered at 20 mL/min over 1 minute to complete one cycle.
Peptides were synthesized on 1% divinyl benzene crosslinked
polystyrene resin. To produce C-terminal carboxamide peptides, 175
mg of MBHA functionalized resin with a stated loading of 1 mmol per
gram was used, and the TFA labile Rink linker was coupled as the
first amino acid. To produce C-terminal hydrazide peptides, 200 mg
of chlorotrityl hydrazide (hydrazide) resin, prepared as below, was
used.
[0157] Non-cysteine containing peptides were cleaved from the resin
and side-chain deprotected by treatment with 2.5% (v/v) water and
2.5% (v/v) TIPS in TFA for two hours at RT. Cysteine containing
peptides were cleaved from the resin and side chain deprotected
with 2.5% (v/v) EDT, 2.5% (v/v) water, and 1% (v/v) TIPS in TFA for
two hours at RT. In all cases, the resin was removed and nitrogen
was used to evaporate the cleavage solution to dryness at RT. The
resulting solids were washed three times with cold diethyl ether,
dissolved in 50% A/50% B (v/v), and lyophilized. Side chain
protection was as follows: Arg(Pbf), Tyr(tBu), Lys(Boc), Asp(OtBu),
Gln(Trt), Ser(tBu), His(Trt), Asn(Trt), Trp(Boc), Glu(OtBu),
Thr(tBu), Cys(Trt).
[0158] For studies conducted with the second generation reactor, a
different resin was used to generate C-terminal hydrazides, because
the Wang resin, used above, was found to promote significant double
incorporation of glycine. We were unable to solve this problem, so
changed resins. The hydrazide resin was produced as follows: 16
grams of chlorotrityl chloride resin with a stated loading of 1.2
mmol/gram were suspended in 150 mL of dry, amine free DMF and
stirred for 15 minutes. To this, a suspension of 25 mL of DIEA, 50
mL of DMF, and 10 mL of anhydrous hydrazine was added drop wise.
During addition, two layers formed and the bottom was added first.
After addition was complete, the mixture was allowed to stir for
one hour, and was then quenched with 50 mL of methanol. The resin
was removed and washed with five 100 mL portions of each of DMF,
water, DMF, methanol, and diethyl ether (for a total wash volume of
2.5 L). The resin was then dried for three hours at reduced
pressure (.apprxeq.5 torr), resulting in a free flowing powder with
lumps. The lumps were gently broken before use, taking care not to
generate fines that could clog the reactor's frit.
[0159] FIGS. 16A-D show crude LCMS chromatograms for ACP(65-74)
synthesized with the second generation protocol at (A) 60.degree.
C. using HATU as an activator, (B) 60.degree. C. using HBTU as an
activator, (C) room temperature using HBTU as an activator, and D)
room temperature synthesis using a comparable manual batch method.
For comparison, FIGS. 16E-H show ACP(65-74) synthesized under
comparable conditions with the first generation reactor and
protocol: (E) 60.degree. C. using HATU as an activator, (F)
60.degree. C. using HBTU as an activator, (G) room temperature
using HBTU as an activator, and (H) room temperature using a
comparable manual batch method. The total ion current is displayed
in each chromatogram.
Example 14
[0160] This example compares the synthesis of ACP(65-74),
conotoxin, HIV-1 protease fragment, and affibody fragments in the
second generation reactor using the synthetic timeline in FIG. 12G
to the synthesis of ACP(65-74), conotoxin, HIV-1 protease fragment,
and affibody fragments in the first generation reactor as described
in Examples 1, 5, and 6. The peptides formed in the second
generation reactor were of comparable quality to the peptides
synthesized in the first generation reactor.
[0161] To compare the performance of the first and second
generation synthesis protocols and reactors, ACP(65-74) was
synthesized with the second generation reactor and cycle with HATU
activation, HBTU activation, and HBTU activation at room
temperature. For comparison, the data in FIG. 4 is reproduced, and
the accelerated room temperature batch experiment was repeated. The
standard second generation syntheses with HBTU and HATU are
slightly worse than the first generation synthesis, but the second
generation RT control has significantly reduced co-eluting Gln
deletion product. The performance of the two protocols is
different, but comparable. Overall, the second generation system is
preferred for its reduced cycle time and vastly improved mechanical
reliability. As of this writing, our lab exclusively uses the
second generation protocol and reactor for peptide synthesis.
[0162] To further compare the performance of the first and second
generation synthesis protocols and reactors, the syntheses of the
HIV-1 protease fragment and the PnI(A10L) Conotoxin were repeated.
The data is shown in FIG. 18, along with the data from FIG. 5 for
comparison. The performance of the two protocols is comparable.
[0163] To validate the performance of the chlorotrityl hydrazide
resin and further explore synthesis with the second generation
reactor and cycle, the synthesis of the affibody fragments was
repeated. The data is shown in FIG. 19, along with the data from
FIG. 7 for comparison. The performance of the two linkers is
comparable when synthesizing fragments without glycine. The
modified Wang resin promotes significant double incorporation of
glycine, which motivated our move to hydrazide resin. In both
cases, pure material suitable for ligation was obtained after
preparative chromatography.
[0164] To further highlight the utility of the second generation
synthesizer, a library of 10 glutathione analogues (see FIG. 20),
several cysteine rich peptides (see FIG. 21), and two biotinylated
protease recognition sites (see FIG. 17) were synthesized. The
glutathione analogues were all produced in one day. In FIG. 21, all
cysteines were acetamidomethyl (Acm) protected, to prevent side
reactions during cleavage and side chain deprotection. Cysteine in
these peptides was activated with 0.190 mL of DIEA rather than
0.157 mL. In all cases, peptides were produced on a 0.2 mmol scale,
the major peak is the desired product, and crude material was
successfully purified in one preparative RP-HPLC step (purified
material is not shown).
[0165] FIG. 17 shows chromatograms of synthesized biotinylated
protease recognition sites. B is .beta.-alanine, K* is biotinylated
lysine, and K' is alloc protected lysine. The alloc protected
peptide was hydrogenated in batch on-resin to give free lysine, and
biotin was coupled in batch. Material eluting after 8 minutes is
non-peptidic.
[0166] FIG. 18 shows chromatograms for the resynthesis of HIV-1
protease fragment and PnI(A10L) conotoxin. Specifically, FIG. 18A-D
show (A) conotoxin synthesized with the second generation reactor
and conditions, (B) HIV-1 PR (81-99) synthesized with the second
generation reactor and conditions, (C) conotoxin synthesized with
the first generation reactor and conditions, and (D) HIV-1 PR
(81-99) synthesized with the first generation reactor and
conditions. Late eluting material was side chain protected
products. Retention times differ slightly because the LC/MS column
was replaced between first and second generation reactor studies.
Material eluting after 15 minutes was non-peptidic.
[0167] FIG. 19 shows chromatograms of affibody fragments. FIGS.
19A-C show fragments synthesized on chlorotrityl hydrazide
functionalized polystyrene with the second generation protocol and
FIGS. 19D-F show fragments synthesized on modified Wang resin with
the first generation protocol.
[0168] FIG. 20 shows chromatograms for a library of glutathione
analogues synthesized in 18 minutes each. E* represents glutamic
acid coupled to the polypeptide through the side chain carboxylate.
All peptides were analyzed under condition 3, and the main peak has
the desired mass.
[0169] FIG. 21 shows chromatograms for several cysteine rich
peptides. All cysteine was Acm protected. Material in FIGS. 21A and
B was analyzed under condition 3, and material in FIG. 21C was
analyzed under condition 4.
Example 15
[0170] This example describes the design and construction of the
automated flow platform.
[0171] The automated platform was designed to perform to the same
standard as the manual system, without the restrictions on cycle
time imposed by a human operator. To accomplish these goals, we
assembled the system shown in FIG. 23A. The automated flow based
platform contained two HPLC pumps, a static mixer, a
microcontroller (not shown), a heat exchanger, a second generation
reactor, and a UV detector amongst other components. The two HPLC
pumps delivered reagents. One HPLC pump delivered solutions of
amino acids and HBTU dissolved in DMF, the other delivered DIEA to
complete activation. Amino acids were stored as 0.4 M solutions
with equimolar HBTU in DMF, and were stable for weeks without the
activating base if stored in sealed reactors.
[0172] A static mixer was used to ensure effective mixing of DIEA
and the amino acid solutions, and the valve positions and pump flow
rates were controlled by an Arduino microcontroller. The static
mixer blended these two fluids, and the heat exchanger, second
generation reactor, and UV detector described above were used
without modification. To increase the residence time between the
static mixer and reactor, an additional six feet of 0.030'' ID
tubing was used. The increased residence time may promote complete
activation, and peptides produced with this extra length of tubing
in the system were observed to be of slightly higher crude purity
than peptides without it.
[0173] All of the valves shown in FIG. 23A were contained in two
manifolds and were actuated by 24 V solenoids. The manifolds were
designed to have minimal internal volume and give zero cross talk
between reagents. One manifold consisted of valves to select one of
five reagents, and one valve to turn the manifold on or off. When
de-energized, the reagent valves selected DMF and the on/off valve
turned the manifold off. These features prevented any incorrect
incorporation of amino acids, minimized the volume of solvent
required to wash the manifold, and prevented siphoning solvent. The
entire flow path was PTFE. Inlet lines were protected by 2 micron
inlet filters.
[0174] The pumps were both Knauer Smartline model 100 with 50
mL/min pump heads. These pumps were selected because they were
readily available and easily controlled with an analog voltage.
However, the pumps had a relatively low pumping capacity and
required about 10 mL of solvent to wash the pump heads. The cycle
time could be further decreased by selecting pumps that have a
higher pumping capacity and require a lower volume of solvent to
wash the pump heads.
[0175] The static mixer was supplied by StaMixCo, and consists of 6
of their smallest elements (6 mm). The heat exchanger, described
previously, was placed between the mixer and second generation
reactor. All three of these elements, and the extra length of
tubing, were placed in a 60.degree. C. water bath. The UV detector
described previously was used to monitor the absorbance of the
waste stream. This device was controlled by an Arduino Mega2560
microcontroller, selected for its simplicity and stability. To
supply a 0-10V analog signal to control the pumps, the Arduino's
0-5 V pulse width modulated outputs were used. The signal was
filtered and amplified to generate a 0-10 V analog control voltage.
To control the valves, the Arduino's digital outputs were used to
actuate power transistors that then supplied 24V DC power from an
external supply. All grounds were common with building ground.
[0176] FIG. 23 shows the automated peptide synthesis platform and
several model peptides synthesized with it. FIG. 23A-E are (A) a
schematic of the device, (B) the synthetic timeline used by this
device to incorporate an amino acid residue every 107 seconds, (C)
a chromatogram showing ALFALFA (SEQ ID NO: 48) assembled in 12
minutes, (D) a chromatogram showing ACP(65-74) assembled in 18
minutes (1,2=Ile deletion, 3=hydrolysis of the C-terminus), and (E)
a chromatogram showing (ALF)7 assembled in 37 minutes. Total ion
chromatograms are shown.
Example 16
[0177] This example describes the synthesis of peptides using the
automated system. The automated cycle time was not limited by the
rate at which a user could complete manual tasks or a syringe pump
could infuse, so the timeline was substantially accelerated (see
FIG. 23B). Two 45 second washes at 50 mL/min (the maximum
available), a seven second coupling, and a ten second deprotection
resulted in the incorporation of an amino acid residue every 107
seconds (1.8 minutes). The coupling time and deprotection time
represent such a small fraction of the total time that the times
were not optimized. However, even faster times can be achieved by
optimizing the coupling time and deprotection time. Faster addition
cycles can also be achieved by eliminating one or more of the wash
steps. Using these cycles, ALFALFA (SEQ ID NO: 48) was produced in
12.5 minutes, ACP(65-74) was produced in 17.8 minutes, and (ALF)7,
a model 21-mer synthesized to demonstrate that the system is
mechanically robust, was produced in 37.5 minutes. The crude
quality of (ALF)7 was nearly identical to a synthesis using the
manual second generation synthesis protocol (see FIG. 24).
[0178] The following control cycle was used to synthesize peptides
with the automated system. The desired solution of amino acid and
activator was selected and delivered at 50 mL/min by the first HPLC
pump. Four seconds later, the second HPLC pump began delivering
DIEA at 9 mL/min. After three more seconds (for a total coupling of
7 seconds) the manifolds selected DMF wash solvent. Sixteen seconds
later, when the amino acid was almost completely washed out of the
manifold and pump, the second HPLC pump was deactivated. After
another 31 seconds (for 45 seconds of total wash time at 50
mL/min), 50% piperidine in DMF was delivered for 10 seconds at 50
mL/min. Finally, DMF was delivered for another 45 seconds at 50
mL/min, completing the final wash and the cycle. Solutions of amino
acid and activator were prepared by dissolving 40 mmol of Na-Fmoc
protected amino acid in 100 mL of 0.4 M HBTU in DMF. Side chain
protection was as follows: Arg(Pbf), Tyr(tBu), Asp(OtBu), Gln(Trt),
Asn(Trt). No correction was made for the change in volume upon
dissolution of the amino acids.
[0179] Three peptides, ALFALFA (SEQ ID NO: 48), ACP(65-74), and
(ALF)7 were produced with the fully automated system. ALFALFA (SEQ
ID NO: 48) was of extremely high crude purity, and ACP(65-74) has
been synthesized many times under many conditions with the manual
system. To compare the automated synthesis of (ALF)7 to the manual
system, (ALF)7 was produced with the second generation reactor and
protocol. The chromatograms are shown in FIG. 24. The quality of
the two crude products is very similar. FIG. 22 shows chromatograms
for (ALF)7 synthesized with (A) the automated 1.8 minute cycle and
(B) manual three minute cycle.
Example 17
[0180] This example describes the synthesis of a peptide using
addition cycles that lacked one or more reagent removal step. In
some cases, as illustrated in this FIG. 24, one or more reagent
removal step is optional and the absence of the removal step does
not negatively affect the performance of subsequent steps and/or
addition cycles.
[0181] Two peptides differing only in the steps included in
addition cycle were synthesized. The sequence of the peptide was
H.sub.2N-CDINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR-CONH.sub.2 (SEQ ID
NO: 35). One peptide was synthesized as described in Example 13 and
served as the control. The addition cycles consisted of amide bond
formation (coupling), removal of coupling reagent (wash), Na Fmoc
removal (deprotection), and removal of the deprotection reagent and
reaction product, piperidine-dibenzofulvene (piperidine-DBF)
(wash). The chromatogram for the synthesized peptide is shown in
FIG. 24A. Another peptide was synthesized as described in Example
13, except the additional cycles did not include the coupling agent
removal step (i.e., coupling agent wash step). The incoming solvent
from the subsequent step (e.g., deprotection step) served to wash
away at least a portion of the coupling reagent and/or reaction
byproducts and the remaining coupling reagent was destroyed by the
excess incoming deprotection reagent. The chromatogram of the
peptide synthesized using addition cycles that lacked a coupling
reagent wash step is shown FIG. 24B. No significant difference was
observed between the peptide lacking the optional wash step and the
peptide that included the optional wash step.
Example 18
[0182] This example describes the synthesis of the 130 residue
DARPin pE59 and the 113 residue Barnase. Each of these proteins was
synthesized from four peptide fragments using ligation chemistry
known to those of ordinary skill in the art and substantially
similar to that described in Example 6 for the affibody.
[0183] The DARPin was synthesized from four fragments, with total
ion chromatograms shown in FIG. 25B-E:
D[1] (H2N-[1Gly-31Gly]-CONHNH2),
D[2] (H2N-[32Cys-64Gly]-CONHNH2),
D[3] (H2N-[65Cys-97Gly]-CONHNH2), and
D[4] (H2N-[98Cys-130Asn]-CONH2).
[0184] These were synthesized using the second generation reactor
described in Example 8, methods described in Example 13, and the
HBTU coupling agent with the following exceptions. All cysteines
were Acm protected.
[0185] D[1] was synthesized in two minutes per residue with two 30
second washes at 40 ml/min. Further, the deprotection was performed
for 20 seconds at 40 ml/min due to difficulties in manually
changing the pump flow rate. It is believed that shorter
deprotections and/or lower flow rate deprotections and/or
deprotections with more dilute deprotection reagent (e.g. 20%
piperidine in DMF) would be effective with this accelerated cycle
and conserve deprotection reagent.
[0186] D[2] and D[3] were synthesized as described in Examples 8
and 13. D[4] was synthesized on aminomethyl-polysyrene resin,
rather than MBHA-polystyrene resin. The resin was prepared as
described in Example 20. 114Ser and 113Ile were incorporated as a
2,2 dimethyl pseudoproline dipeptide, and coupled for 10 minutes by
pausing the addition cycle after complete delivery of the coupling
reagent. It is believed that this pause is unnecessary. 116Asp was
incorporated with 3-methyl pentyl ester side chain protection,
rather than standard t-butyl ester side chain protection. Finally,
deprotections were performed for 20 seconds at 20 ml/min with 20%
piperidine and 0.1M HOBt in DMF.
[0187] Barnase was synthesized from four fragments, with total ion
chromatograms shown in FIG. 26B-E:
B[1] (H2N-[1Gly-13Val]-CONHNH2)
B[2] (H2N-[14Cys-39Val]-CONHNH2).
B[3] (H2N-[40Cys-76Glu]-CONHNH2) and
B[4] (H2N-[74Cys-113Arg]-CONH2).
[0188] These were synthesized using the reactor described in
Example 8, methods described in Example 13, and the HATU coupling
agent with the following exceptions. All peptides were synthesized
using 20% piperidine in DMF as the deprotection reagent. All
cysteines were Acm protected, and 76Glu was incorporated as the
cyclohexyl ester. For the synthesis of B[1], the c-terminal valine
was coupled to the resin for 10 minutes by pausing the addition
cycle after delivery of the valine coupling reagent. It is believed
that a shorter coupling and/or a coupling at higher temperature can
be used, however it was found that the 30 second coupling at
60.degree. C. described in Example 13 was not adequate to
quantitatively bond sterically hindered valine to sterically
hindered trityl hydrazide resin.
[0189] For the synthesis of B[2], the c-terminal valine was coupled
to the resin for 10 minutes, as in B[1], and 33Ala was incorporated
with hydroxy methoxy benzyl (HMB) backbone protection. This residue
was activated with DCC/HOBt rather than HATU and coupled for 25
minutes by pausing the amino acid addition cycle for 25 minutes
after complete delivery of the coupling reagent. The subsequent
glutamic acid was coupled for 30 minutes by pausing the amino acid
addition cycle for 30 minutes after complete delivery of the
coupling reagent. It is believed that incorporation of HMB
protected amino acids can be accelerated by operating at higher
temperature.
B3 was synthesized as described above and in Examples 8 and 13. B4
was synthesized on Rink-PEG resin supplied by ChemMatrix, and 90Arg
was activated with DCC/HOBt rather than HATU.
[0190] After preparation of the crude protein fragments, they were
purified by RP-HPLC using methods known to those of ordinary skill
in the art and substantially similar to those described in Example
12. The purified fragments were then ligated according to the
schemes in FIGS. 25A and 26A to afford the full length proteins
shown in FIGS. 25F and 26F, respectively. Ligation was performed
with methods known to those of ordinary skill in the art, following
the procedure of Liu and co-workers with minor modifications.
Example 19
[0191] This example describes the synthesis of the A and B chains
of human insulin and an evolved integrin binding scaffold based on
the EETI-II trypsin inhibitor. Total ion chromatograms of the crude
synthetic products are shown in FIG. 27A-C, respectively. All of
these peptides were synthesized using amino methyl functionalized
polystyrene instead of MBHA functionalized polystyrene. The amino
methyl functionalized polystyrene was prepared using methods known
to those of ordinary skill in the art and as described below in
Example 20.
[0192] The A chain of human insulin was synthesized using the
second generation reactor described in Example 8 and the methods
described in Example 13, except the synthesis was conducted at
85.degree. C. and all cysteines were Acm protected. The B chain of
human insulin was synthesized using the reactor described in
Example 8 and the methods described in Example 13, except the
synthesis was conducted at 85.degree. C. The integrin binding
scaffold was synthesized using the reactor described in Example 8
and the methods described in Example 13, except all chiral amino
acids were of inverted chirality (i.e. dextrorotatory).
[0193] While several embodiments of the present invention have been
described and illustrated herein, those of ordinary skill in the
art will readily envision a variety of other means and/or
structures for performing the functions and/or obtaining the
results and/or one or more of the advantages described herein, and
each of such variations and/or modifications is deemed to be within
the scope of the present invention. More generally, those skilled
in the art will readily appreciate that all parameters, dimensions,
materials, and configurations described herein are meant to be
exemplary and that the actual parameters, dimensions, materials,
and/or configurations will depend upon the specific application or
applications for which the teachings of the present invention
is/are used.
Example 20
[0194] This example describes a procedure for preparation of amino
methyl resin used in Examples 18 and 19.
[0195] To prepare amino methyl resin, 25 g of Bio-Rad, Bio-beads
S-X1 (styrene-divinylbenzene copolymer, 1% crosslinkage) and 4.9 g
(27.6 mmol) of N-hydroxymethylphthalimide were added to 450 mL of
DCM in a 1 L round bottom flask. To this, 50 mL of methanesulfonic
acid was added and the reaction was stirred gently for 5 hours at
room temperature. After 5 hours of stirring, the slurry was
transferred to a coarse fritted glass funnel and washed with DCM
(1500-2000 mL) and ethanol (1500 mL). The resin was then dried
under vacuum for 1 hour. After drying, the phthalimidomethyl-resin
was transferred to a 500 mL round bottom flask and suspended in a
200 mL solution of hydrazine monohydrate (20% v/v) in absolute
ethanol. A reflux condenser was attached and the solution was
refluxed gently for at least 8 hours. The resulting gelatinous
material was transferred hot to a glass fritted funnel and washed
with boiling ethanol (1000-2000 mL) and then hot methanol (1000 mL)
in order to completely wash away the white phthalhydrazide
precipitate. The resin was then washed with DMF (800 mL), DCM (800
mL), 10% (v/v) DIEA in DMF (500 mL), DMF (600 mL), and DCM (1000
mL). The resin was then dried under vacuum and determined to have a
loading of 1.2 mmol/g.
[0196] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. It is, therefore, to be understood that the foregoing
embodiments are presented by way of example only and that, within
the scope of the appended claims and equivalents thereto, the
invention may be practiced otherwise than as specifically described
and claimed. The present invention is directed to each individual
feature, system, article, material, and/or method described herein.
In addition, any combination of two or more such features, systems,
articles, materials, and/or methods, if such features, systems,
articles, materials, and/or methods are not mutually inconsistent,
is included within the scope of the present invention.
[0197] The indefinite articles "a" and "an," as used herein in the
specification and in the claims, unless clearly indicated to the
contrary, should be understood to mean "at least one."
[0198] The phrase "and/or," as used herein in the specification and
in the claims, should be understood to mean "either or both" of the
elements so conjoined, i.e., elements that are conjunctively
present in some cases and disjunctively present in other cases.
Other elements may optionally be present other than the elements
specifically identified by the "and/or" clause, whether related or
unrelated to those elements specifically identified unless clearly
indicated to the contrary. Thus, as a non-limiting example, a
reference to "A and/or B," when used in conjunction with open-ended
language such as "comprising" can refer, in one embodiment, to A
without B (optionally including elements other than B); in another
embodiment, to B without A (optionally including elements other
than A); in yet another embodiment, to both A and B (optionally
including other elements); etc.
[0199] As used herein in the specification and in the claims, "or"
should be understood to have the same meaning as "and/or" as
defined above. For example, when separating items in a list, "or"
or "and/or" shall be interpreted as being inclusive, i.e., the
inclusion of at least one, but also including more than one, of a
number or list of elements, and, optionally, additional unlisted
items. Only terms clearly indicated to the contrary, such as "only
one of" or "exactly one of," or, when used in the claims,
"consisting of," will refer to the inclusion of exactly one element
of a number or list of elements. In general, the term "or" as used
herein shall only be interpreted as indicating exclusive
alternatives (i.e. "one or the other but not both") when preceded
by terms of exclusivity, such as "either," "one of," "only one of,"
or "exactly one of." "Consisting essentially of," when used in the
claims, shall have its ordinary meaning as used in the field of
patent law.
[0200] As used herein in the specification and in the claims, the
phrase "at least one," in reference to a list of one or more
elements, should be understood to mean at least one element
selected from any one or more of the elements in the list of
elements, but not necessarily including at least one of each and
every element specifically listed within the list of elements and
not excluding any combinations of elements in the list of elements.
This definition also allows that elements may optionally be present
other than the elements specifically identified within the list of
elements to which the phrase "at least one" refers, whether related
or unrelated to those elements specifically identified. Thus, as a
non-limiting example, "at least one of A and B" (or, equivalently,
"at least one of A or B," or, equivalently "at least one of A
and/or B") can refer, in one embodiment, to at least one,
optionally including more than one, A, with no B present (and
optionally including elements other than B); in another embodiment,
to at least one, optionally including more than one, B, with no A
present (and optionally including elements other than A); in yet
another embodiment, to at least one, optionally including more than
one, A, and at least one, optionally including more than one, B
(and optionally including other elements); etc.
[0201] In the claims, as well as in the specification above, all
transitional phrases such as "comprising," "including," "carrying,"
"having," "containing," "involving," "holding," and the like are to
be understood to be open-ended, i.e., to mean including but not
limited to. Only the transitional phrases "consisting of" and
"consisting essentially of" shall be closed or semi-closed
transitional phrases, respectively, as set forth in the United
States Patent Office Manual of Patent Examining Procedures, Section
2111.03.
Sequence CWU 1
1
4817PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified with Fmoc 1Ala Leu Phe Ala
Leu Phe Ala1 525PRTArtificial SequenceSynthetic Polypeptide 2Leu
Tyr Arg Ala Gly1 5310PRTArtificial SequenceSynthetic Polypeptide
3Val Gln Ala Ala Ile Asp Tyr Ile Asn Gly1 5 10416PRTArtificial
SequenceSynthetic Polypeptide 4Gly Cys Cys Ser Leu Pro Pro Cys Ala
Leu Asn Asn Pro Asp Tyr Cys1 5 10 15519PRTArtificial
SequenceSynthetic Polypeptide 5Pro Val Asn Ile Ile Gly Arg Asn Leu
Leu Thr Gln Ile Gly Cys Thr1 5 10 15Leu Asn Phe627PRTArtificial
SequenceSynthetic Polypeptide 6Val Asp Asn Lys Phe Asn Lys Glu Gln
Gln Asn Ala Phe Tyr Glu Ile1 5 10 15Leu His Leu Pro Asn Leu Asn Glu
Glu Gln Arg 20 25711PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified with Thz 7Ala Phe Ile Gln
Ser Leu Lys Asp Asp Pro Ser1 5 10819PRTArtificial SequenceSynthetic
Polypeptide 8Cys Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn
Asp Ala Gln1 5 10 15Ala Pro Lys927PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified with Fmoc 9Val Asp Asn Lys
Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile1 5 10 15Leu His Leu
Pro Asn Leu Asn Glu Glu Gln Arg 20 251033PRTArtificial
SequenceSynthetic PolypeptideMISC_FEATURE(1)..(1)Modified with
Bocmisc_feature(33)..(33)Xaa can be any naturally occurring amino
acid 10Gly Gly Gly Gly Gly Val Asp Asn Lys Phe Asn Lys Glu Gln Gln
Asn1 5 10 15Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu
Gln Arg 20 25 30Xaa1111PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified with Fmoc and Thz 11Ala Phe
Ile Gln Ser Leu Lys Asp Asp Pro Ser1 5 101212PRTArtificial
SequenceSynthetic PolypeptideMISC_FEATURE(1)..(1)Modified with Boc
and Thzmisc_feature(12)..(12)Xaa can be any naturally occurring
amino acid 12Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Xaa1 5
101319PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified with Fmoc 13Cys Ser Ala Asn
Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln1 5 10 15Ala Pro
Lys1419PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Modified with Boc 14Cys Ser Ala Asn
Leu Leu Ala Glu Ala Lys Lys Leu Asn Asp Ala Gln1 5 10 15Ala Pro
Lys1539PRTArtificial SequenceSynthetic Polypeptide 15Val Asp Asn
Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile1 5 10 15Leu His
Leu Pro Asn Leu Asn Glu Glu Gln Arg Cys Ala Phe Ile Gln 20 25 30Ser
Leu Lys Asp Asp Pro Ser 351624PRTArtificial SequenceSynthetic
Polypeptide 16Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly
Thr Ser Gly1 5 10 15Arg Gly Gly Leu Gly Gly Gln Gly
201711PRTArtificial SequenceSynthetic Polypeptide 17Gly Gly Gly Gly
Gly Ala Arg Leu Leu Arg Leu1 5 101819PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(6)..(6)Xaa is
bAlamisc_feature(15)..(15)Xaa is bAlamisc_feature(18)..(18)Xaa is
bAlaMISC_FEATURE(19)..(19)Alloc protected 18Gly Gly Gly Gly Gly Xaa
Leu Val Pro Arg Gly Ser Gly Gly Xaa Gly1 5 10 15Gly Xaa
Lys1919PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(13)..(13)Xaa is
bAlamisc_feature(16)..(16)Xaa is
bAlaMISC_FEATURE(19)..(19)Biotinylated 19Gly Gly Gly Gly Gly Glu
Asn Leu Tyr Phe Gln Ser Xaa Gly Gly Xaa1 5 10 15Gly Gly
Lys2012PRTArtificial SequenceSynthetic Polypeptide 20Cys Ala Phe
Ile Gln Ser Leu Lys Asp Asp Pro Ser1 5 10216PRTArtificial
SequenceSynthetic Polypeptide 21Glu Cys Gly Gly Leu Leu1
5226PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Glutamic acid coupled to polypeptide
through side chain carboxylate 22Glu Cys Gly Gly Leu Leu1
5236PRTArtificial SequenceSynthetic Polypeptide 23Asp Cys Gly Pro
Leu Leu1 5246PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Glutamic acid coupled to polypeptide
through side chain carboxylate 24Glu Cys Gly Pro Leu Leu1
5257PRTArtificial SequenceSynthetic Polypeptide 25Asp Glu Cys Gly
Lys Leu Leu1 5266PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Glutamic acid coupled to polypeptide
through side chain carboxylate 26Glu Cys Gly Lys Leu Leu1
5276PRTArtificial SequenceSynthetic Polypeptide 27Asn Cys Gly His
Leu Leu1 5286PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Glutamic acid coupled to polypeptide
through side chain carboxylate 28Glu Cys Gly His Leu Leu1
5296PRTArtificial SequenceSynthetic Polypeptide 29Gln Cys Gly Leu
Leu Leu1 5306PRTArtificial SequenceSynthetic
PolypeptideMISC_FEATURE(1)..(1)Glutamic acid coupled to polypeptide
through side chain carboxylate 30Glu Cys Gly Leu Leu Leu1
53117PRTArtificial SequenceSynthetic Polypeptide 31Cys Ala Cys Gly
Ala Leu Tyr Lys Lys Gly Ser Phe Ala Gly Cys Ala1 5 10
15Cys3217PRTArtificial SequenceSynthetic Polypeptide 32Cys Ala Gly
Cys Ala Leu Tyr Lys Lys Gly Ser Phe Ala Cys Gly Ala1 5 10
15Cys3317PRTArtificial SequenceSynthetic Polypeptide 33Cys Cys Ala
Gly Ala Leu Tyr Lys Lys Gly Ser Phe Ala Gly Ala Cys1 5 10
15Cys3421PRTArtificial SequenceSynthetic Polypeptide 34Ala Leu Phe
Ala Leu Phe Ala Leu Phe Ala Leu Phe Ala Leu Phe Ala1 5 10 15Leu Phe
Ala Leu Phe 203537PRTArtificial SequenceSynthetic Polypeptide 35Cys
Asp Ile Asn Tyr Thr Ser Gly Phe Arg Asn Ser Asp Arg Ile Leu1 5 10
15Tyr Ser Ser Asp Trp Leu Ile Tyr Lys Thr Thr Asp His Tyr Gln Thr
20 25 30Phe Thr Lys Ile Arg 3536130PRTArtificial SequenceSynthetic
Polypeptidemisc_feature(28)..(28)Xaa is Nle 36Gly Gly Gly Gly Gly
Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala1 5 10 15Arg Ala Gly Gln
Asp Asp Glu Val Arg Ile Leu Xaa Ala Asn Gly Ala 20 25 30Asp Val Asn
Ala Leu Asp Glu Asp Gly Leu Thr Pro Leu His Leu Ala 35 40 45Ala Gln
Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr Gly 50 55 60Ala
Asp Val Asn Ala Glu Asp Asn Phe Gly Ile Thr Pro Leu His Leu65 70 75
80Ala Ala Ile Arg Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His
85 90 95Gly Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe
Asp 100 105 110Ile Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile
Leu Gln Lys 115 120 125Leu Asn 1303731PRTArtificial
SequenceSynthetic Polypeptidemisc_feature(28)..(28)Xaa is Nle 37Gly
Gly Gly Gly Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala1 5 10
15Arg Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Xaa Ala Asn Gly 20 25
303833PRTArtificial SequenceSynthetic Polypeptide 38Cys Asp Val Asn
Ala Leu Asp Glu Asp Gly Leu Thr Pro Leu His Leu1 5 10 15Ala Ala Gln
Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Tyr 20 25
30Gly3933PRTArtificial SequenceSynthetic Polypeptide 39Cys Asp Val
Asn Ala Glu Asp Asn Phe Gly Ile Thr Pro Leu His Leu1 5 10 15Ala Ala
Ile Arg Gly His Leu Glu Ile Val Glu Val Leu Leu Lys His 20 25
30Gly4033PRTArtificial SequenceSynthetic Polypeptide 40Cys Asp Val
Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile1 5 10 15Ser Ile
Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Leu 20 25
30Asn41113PRTArtificial SequenceSynthetic Polypeptide 41Gly Gly Gly
Ala Gln Val Ile Asn Thr Phe Asp Gly Val Ala Asp Tyr1 5 10 15Leu Gln
Thr Tyr His Lys Leu Pro Asp Asn Tyr Ile Thr Lys Ser Glu 20 25 30Ala
Gln Ala Leu Gly Trp Val Ala Ser Lys Gly Asn Leu Ala Asp Val 35 40
45Ala Pro Gly Lys Ser Ile Gly Gly Asp Ile Phe Ser Asn Arg Glu Gly
50 55 60Lys Leu Pro Gly Lys Ser Gly Arg Thr Trp Arg Glu Ala Asp Ile
Asn65 70 75 80Tyr Thr Ser Gly Phe Arg Asn Ser Asp Arg Ile Leu Tyr
Ser Ser Asp 85 90 95Trp Leu Ile Tyr Lys Thr Thr Asp His Tyr Gln Thr
Phe Thr Lys Ile 100 105 110Arg4213PRTArtificial SequenceSynthetic
Polypeptide 42Gly Gly Gly Ala Gln Val Ile Asn Thr Phe Asp Gly Val1
5 104326PRTArtificial SequenceSynthetic Polypeptide 43Cys Asp Tyr
Leu Gln Thr Tyr His Lys Leu Pro Asp Asn Tyr Ile Thr1 5 10 15Lys Ser
Glu Ala Gln Ala Leu Gly Trp Val 20 254437PRTArtificial
SequenceSynthetic Polypeptide 44Cys Ser Lys Gly Asn Leu Ala Asp Val
Ala Pro Gly Lys Ser Ile Gly1 5 10 15Gly Asp Ile Phe Ser Asn Arg Glu
Gly Lys Leu Pro Gly Lys Ser Gly 20 25 30Arg Thr Trp Arg Glu
354521PRTArtificial SequenceSynthetic Polypeptide 45Gly Ile Val Glu
Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu1 5 10 15Glu Asn Tyr
Cys Asn 204630PRTArtificial SequenceSynthetic Polypeptide 46Phe Val
Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr1 5 10 15Leu
Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25
304733PRTArtificial SequenceSynthetic Polypeptide 47Gly Cys Asx Arg
Pro Arg Gly Asp Asn Pro Pro Leu Asx Cys Ser Gln1 5 10 15Asp Ser Asp
Cys Leu Ala Gly Cys Val Cys Gly Pro Asn Gly Phe Cys 20 25
30Gly487PRTArtificial SequenceSynthetic Polypeptide 48Ala Leu Phe
Ala Leu Phe Ala1 5
* * * * *