Convenient synthesis of collagen-related tripeptides for segment condensation

Article abstract of DOI:10.1002/bip.22700

Chromatography is a common step in the solution-phase synthesis of typical peptides, as well as peptide fragments for subsequent coupling on a solid support. Combining known reagents that form readily separable byproducts is shown to eliminate this step, which wastes time and other resources. Specifically, activating carboxyl groups with isobutyl chloroformate or as pentafluorophenyl esters and using N-methyl morpholine as a base enable chromatography-free synthetic routes in which peptide products are isolated from byproducts by facile evaporation, extraction, and trituration. This methodology was used to access tripeptides related to collagen, such as Fmoc-Pro-Pro-Gly-OH and Fmoc-Pro-Hyp(tBu)-Gly-OH, in a purity suitable for solid-phase segment condensation to form collagen mimetic peptides.

Full text of DOI:10.1002/bip.22700

Convenient Synthesis of Collagen-Related Tripeptides
for Segment Condensation
Aubrey J. Ellison,1 Brett VanVeller,1,* and Ronald T. Raines1,2
Additional Supporting Information may be found in the online version of this article.
Correspondence to: Ronald T. Raines, Department of Biochemistry, University of Wisconsin–
Madison, 433 Babcock Drive, Madison, WI 53706ꢀ1544. Eꢀmail: rtraines@wisc.edu
1Department of Chemistry, University of Wisconsin–Madison, 1101 University Avenue, Madison,
WI 53706ꢀ1322
*Present address: Department of Chemistry, Iowa State University, Ames, IA 50011ꢀ3111
2Department of Biochemistry, University of Wisconsin–Madison, 433 Babcock Drive, Madison,
WI 53706ꢀ1544
Contract grant sponsor: National Institutes of Health
Contract grant numbers: R01 AR044276, T32 GM008505, and P41 GM103399
Contract grant sponsor: Canadian Institute of Health Research
Contract grant number: 289613
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
‘Accepted Article’, doi: 10.1002/bip.22700
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Abstract
Chromatography is a common step in the solutionꢀphase synthesis of typical peptides, as well as
peptide fragments for subsequent coupling on a solid support. Combining known reagents that
form readily separable byproducts is shown to eliminate this step, which wastes time and other
resources. Specifically, activating carboxyl groups with isobutyl chloroformate or as
pentafluorophenyl esters and using Nꢀmethyl morpholine as a base enable chromatographyꢀfree
synthetic routes in which peptide products are isolated from byproducts by facile evaporation,
extraction, and trituration. This methodology was used to access tripeptides related to collagen,
such as FmocꢀProꢀProꢀGlyꢀOH and FmocꢀProꢀHyp(tBu)ꢀGlyꢀOH, in a purity suitable for solidꢀ
phase segment condensation to form collagen mimetic peptides.
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INTRODUCTION
Synthetic peptides have been the basis for transformative advances in medicine, as well as the
creation of new catalysts and materials.1ꢀ8 Peptides are synthesized either in solution, as
pioneered by Emil Fischer at the beginning of the 20th century,9 or on a solid support, as
developed by Bruce Merrifield in the 1960s.10 Drawbacks occur with both methods. The
synthesis of peptides in solution is plagued by challenging purification steps and other issues.11
Solidꢀphase peptide synthesis (SPPS) facilitates purification12,13 and continues to advance,14ꢀ18
though overall yields necessarily diminish with each coupling step in the linear route and are
intrinsically low for some sequences.19,20
A convergent strategy in which protected peptide fragments made in solution are coupled on
a solid support offers an attractive alternative.21,22,14 Such “segment condensation” requires fewer
couplings, increasing efficiency and reducing waste. In part, however, the use of segment
condensation on a solid support merely displaces inefficiency and waste to the solution phase,
and its requisite chromatographic purification.
Our research group and many others are interested in collagen mimetic peptides (CMPs).23ꢀ25
CMPs have been used to reveal the structure of the collagen triple helix26,27 along with the forces
that underlie that structure,28 and have been of particular relevance in biomedicine and materials
science.29ꢀ35 Like collagen itself, CMPs are composed of XaaꢀYaaꢀGly triplets. This invariant
repetition makes CMPs ideal for synthesis by segment condensation. These factors motivated us
to seek synthetic routes to CMPs that avoid cumbersome purification steps.
Typical CMPs comprise 21–30 residues. The component XaaꢀYaaꢀGly triplets for segment
condensation are made by using solutionꢀphase methods.36ꢀ39 In our experience,40ꢀ43 the synthesis
of tripeptides with common reagents requires chromatography on a column of silica gel (or
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crystallization, which can be idiosyncratic) to obtain products of adequate purity from each
coupling step and the final Cꢀterminal deprotection step. Purification by chromatography is not
only laborious, but also requires the use of chromatographic media and solvents that are typically
discarded as waste. Finally, chromatography of longer peptides or on a large scale suffers from
diminished resolution of products. Here, we report on methodology for the chromatographyꢀfree
synthesis of collagenꢀrelated tripeptides that are poised for SPPS. Our methods are efficient and
scalable, and could be applicable to other peptide fragments.
MATERIALS AND METHODS
General
Pentafluorophenyl (Pfp) esters44 and other amino acid derivatives were from ChemꢀImpex
International (Wood Dale, IL). Isobutyl chloroformate45 and all other reagents were from Sigma–
Aldrich (St. Louis, MO) and were used without further purification. Tetrahydrofuran (THF) and
N,Nꢀdimethylformamide (DMF) were dried with a Glass Contour system from Pure Process
Technology (Nashua, NH). In addition, DMF was passed through an associated isocyanate
“scrubbing” column to remove any amines.
Volatile Removal
The phrase “concentrated under reduced pressure” refers to the removal of solvents and other
volatile materials with a rotary evaporator at water aspirator pressure (<20 torr) while
maintaining a water bath below 40 °C. The phrase “volatiles were removed under reduced
pressure” refers to the removal of solvents and other volatile materials with a rotary evaporator at
vacuumꢀpump pressure (~2 torr) while maintaining a water bath below 40 °C.
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Trituration
Trituration was done by the “solvent swap” method. Briefly, a compound was dissolved in a
mixture of two solvents, one being a more volatile solvent in which the compound has good
solubility (e.g., DCM), the other (the “antiꢀsolvent”) being a less volatile solvent in which the
compound has poor solubility (e.g., hexanes). The resulting solution was concentrated under
reduced pressure to remove the more volatile solvent. The solids were isolated from the slurry by
filtration through a Büchner funnel, and the filter cake was washed with the antiꢀsolvent.
Analysis of Peptides from Solution-Phase Synthesis
Molecular mass was determined at high resolution by electrospray ionization (ESI) mass
spectrometry with an LCT instrument from Waters (Milford, MA).
1H and 13C NMR spectra were acquired with an Avance III 400 spectrometer or Avance III
500I spectrometer equipped with a cryogenic probe from Bruker (Billerica, MA). Chemical
shifts are reported in units of δ (ppm) relative to tetramethylsilane as the internal standard. In the
spectrum of each compound herein, multiple conformers are apparent due to cis−trans isomerism
around amide and/or carbamide bonds.
Peptide purity was assessed by reverseꢀphase chromatography with a Discovery BIO Wide
Pore C5ꢀ5 column from Supelco (Bellefonte, PA). The column was eluted with 5–95% v/v B
over 10 min, 95% v/v B for 2 min, 95–5% over 2 min, and 5% v/v B for 5 min (A: H2O
containing 0.1% v/v formic acid; B: acetonitrile containing 0.1% v/v formic acid). Absorbance at
254 nm and molecular mass were monitored with an LCMSꢀ2020 instrument from Shimadzu
(Kyoto, Japan).
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Solid-Phase Peptide Synthesis
Solidꢀphase peptide synthesis was performed at the University of Wisconsin–Madison
Biotechnology Center with a Prelude peptide synthesizer from Protein Technologies (Tucson,
AZ). Synthetic peptide was purified by HPLC with an LCꢀ20 instrument from Shimadzu.
Molecular mass was determined by matrixꢀassisted laser desorption/ionization–timeꢀofꢀflight
(MALDI–TOF) mass spectrometry on an αꢀcyanoꢀ4ꢀhydroxycinnamic acid matrix with a
Voyager DEꢀPro instrument at the Biophysics Instrumentation Facility at the University of
Wisconsin–Madison.
Synthesis of Cbz-Pro-Gly-OtBu
CbzꢀProꢀOH (3.01 g, 12 mmol) and NMM (4.0 mL, 36 mmol) were dissolved in 200 mL THF,
and the resulting solution was cooled to –78 °C in a dry ice/acetone bath. Isobutyl chloroformate
(1.5 mL, 12 mmol) was added dropwise, and a white precipitate (NMM·HCl) formed. Solid
HClꢁGlyꢀOtBu (2.02 g, 12 mmol) was added in one portion, and the reaction mixture was
allowed to warm to room temperature and then stirred for 12 h. The precipitate was removed by
filtration, and the solution was concentrated under reduced pressure. The residue was dissolved
in EtOAc, and the resulting solution was washed successively with 0.10 M KHSO4 (2×) and
saturated aqueous NaHCO3 (2×). The organic layer was dried over Na2SO4(s), filtered, and
volatiles were removed under reduced pressure to give CbzꢀProꢀGlyꢀOtBu (4.01 g, 92%) as a
pale yellow solid, which was judged to be 99% pure by LCMS. HRMSꢀESI (m/z): [M + H]+
calcd, 363.1915; found, 363.1909. Integration of signals for the N–H proton revealed that the
cis:trans ratio of the prolyl peptide bond was 1.0:1.5. 1H NMR (500 MHz, CDCl3, δ): 7.42–7.28
(m, 5H), 5.32–4.99 (m, 2H), 4.56–4.22 (m, 1H), 3.93 (dt, J = 16.0, 8.8 Hz, 2H), 3.66–3.39 (m,
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2H), 2.43–2.11 (m, 2H), 2.03–1.80 (m, 3H), 1.46 (s, 9H). 13C NMR (126 MHz, CDCl3, δ):
page 7
171.75, 168.75, 136.38, 128.51, 128.09, 127.94, 82.08, 67.33, 60.57, 47.08, 42.09, 28.71, 28.05,
24.52.
Synthesis of Fmoc-Pro-Pro-Gly-OtBu
A suspension of CbzꢀProꢀGlyꢀOtBu (4.01 g, 11 mmol) and Pd/C (1.17 g, 10% w/w) in 25 mL of
MeOH was stirred under an atmosphere of H2(g) for 16 h at room temperature. Any insoluble
material was removed by filtration, and volatiles were removed under reduced pressure. (The
weight of the crude intermediate was 2.48 g.) The residue was dissolved in 35 mL of THF, and
NMM (3.47 mL, 32 mmol) and FmocꢀProꢀOPfp (4.00 g, 7.9 mmol) were added to the resulting
solution. After stirring for 16 h, volatiles were removed under reduced pressure. The residue was
dissolved in EtOAc, and the resulting solution was washed successively with 0.10 M KHSO4
(2×) and saturated aqueous NaHCO3 (2×). The organic layer was dried over Na2SO4(s), filtered,
and concentrated under reduced pressure. The residue was dissolved in the minimum volume of
DCM and triturated with hexanes. To enhance its solidity, material insoluble in hexanes was
dissolved in methanol, and volatiles were removed under reduced pressure to give FmocꢀProꢀ
ProꢀGlyꢀOtBu (4.44 g, 74%) as an offꢀwhite solid, which was judged to be 94% pure by LCMS.
HRMSꢀESI (m/z): [M + H]+ calcd, 548.2756; found, 548.2742. 1H NMR (500 MHz, CDCl3, δ):
7.81–7.72 (m, 2H), 7.66–7.52 (m, 2H), 7.40 (t, J = 7.5 Hz, 2H), 7.31 (tt, J = 7.4, 1.3 Hz, 2H),
4.76–4.14 (m, 5H), 4.04–3.34 (m, 6H), 2.42–1.78 (m, 8H), 1.54–1.29 (m, 9H). 13C NMR (126
MHz, CDCl3, δ): 171.62, 155.21, 144.28, 143.97, 141.43, 127.81, 127.20, 127.15, 125.38,
125.24, 125.18, 120.12, 120.09, 82.10, 81.68, 77.16, 67.95, 67.69, 61.17, 59.97, 59.65, 58.48,
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57.76, 47.33, 47.25, 47.17, 46.99, 42.33, 42.10, 29.57, 28.16, 28.14, 28.07, 27.24, 25.39, 24.88,
24.50, 23.18.
Synthesis of Fmoc-Pro-Pro-Gly-OH
FmocꢀProꢀProꢀGlyꢀOtBu (4.44 g, 8.1 mmol) was dissolved in 12 mL 4 M HCl in dioxane, and
the resulting solution was stirred for 14 h at room temperature. The reaction mixture was
concentrated under reduced pressure. The residue was dissolved in the minimum volume of
DCM and triturated with hexanes. To enhance its solidity, material insoluble in hexanes was
dissolved in methanol, and volatiles were removed under reduced pressure to give FmocꢀProꢀ
ProꢀGlyꢀOH (3.57 g, 90%) as an offꢀwhite solid, which was judged to be 85% pure by LCMS.
1H NMR (500 MHz, CDCl3, δ): 7.75 (ddd, J = 10.3, 7.2, 4.1 Hz, 2H), 7.65–7.49 (m, 2H), 7.43–
7.36 (m, 2H), 7.30 (ttd, J = 7.4, 3.1, 1.4 Hz, 2H), 4.73–4.49 (m, 1H), 4.49–4.18 (m, 3H), 4.18–
3.79 (m, 2H), 3.79–3.64 (m, 2H), 3.64–3.50 (m, 2H), 2.60–1.65 (m, 8H).13C NMR (126 MHz,
CDCl3, δ): 172.49, 172.24, 172.10, 171.91, 171.85, 171.80, 171.69, 155.21, 154.49, 144.28,
144.06, 143.98, 143.72, 143.57, 141.29, 141.25, 141.23, 127.77, 127.72, 127.69, 127.59, 127.08,
127.04, 125.21, 125.07, 124.78, 124.61, 119.96, 119.77, 67.93, 67.70, 66.28, 60.99, 60.06, 59.86,
59.70, 58.75, 58.44, 57.67, 52.27, 47.58, 47.25, 47.16, 47.01, 46.90, 41.33, 41.15, 31.84, 30.15,
29.34, 29.29, 27.68, 27.49, 27.16, 25.17, 25.02, 24.67, 24.39, 23.08, 22.02.
Synthesis of Cbz-D-Pro-Gly-OtBu
CbzꢀDꢀProꢀOH (3.02 g, 12 mmol) and NMM (4.0 mL, 36 mmol) were dissolved in 200 mL THF,
and the resulting solution was cooled to –78 °C in a dry ice/acetone ice bath. Isobutyl
chloroformate (1.5 mL, 12 mmol) was added dropwise and a white precipitate (NMM·HCl)
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formed. Solid HClꢁGlyꢀOtBu (2.04 g, 12 mmol) was added in one portion, and the reaction
mixture was allowed to warm to room temperature and then stirred for 12 h. The precipitate was
removed by filtration, and the solution was concentrated under reduced pressure. The residue
was dissolved in EtOAc, and the resulting solution was washed successively with 0.10 M
KHSO4 (2×) and saturated aqueous NaHCO3 (2×). The organic layer was dried over Na2SO4(s),
filtered, and volatiles were removed under reduced pressure to give CbzꢀDꢀProꢀGlyꢀOtBu (4.27
g, 97%) as a pale yellow solid, which was judged to be 91% pure by LCMS. HRMSꢀESI (m/z):
[M + H]+ calcd, 363.1915; found, 363.1907. Integration of signals for the N–H proton revealed
that the cis:trans ratio of the prolyl peptide bond was 1.0:1.7. 1H NMR (400 MHz, CDCl3, δ):
7.45–7.30 (m, 5H), 5.29–5.03 (m, 2H), 4.37 (d, J = 22.7 Hz, 1H), 4.06–3.81 (m, 2H), 3.66–3.36
(m, 2H), 2.43–2.08 (m, 1H), 2.02–1.84 (m, 3H), 1.46 (s, 9H).13C NMR (126 MHz, CDCl3, δ):
172.30, 171.71, 168.75, 156.15, 136.36, 128.51, 128.10, 127.96, 82.12, 77.28, 77.03, 67.35,
60.90, 60.58, 47.53, 47.09, 46.93, 42.10, 41.68, 31.06, 29.72, 28.64, 28.05, 24.53, 23.63, 19.03.
Synthesis of Fmoc-D-Pro-D-Pro-Gly-OtBu
A suspension of CbzꢀDꢀProꢀGlyꢀOtBu (4.267 g, 12 mmol) and Pd/C (1.25 g, 10% w/w) in 25 mL
of MeOH was stirred under an atmosphere of H2(g) for 18 h at room temperature. Any insoluble
material was removed by filtration, and volatiles were removed under reduced pressure. The
residue was dissolved in 80 mL of 5:3 THF/DMF. NMM (3.87 mL, 35 mmol), HBTU (4.45 g,
12 mmol), and FmocꢀDꢀProꢀOH (3.97 g 12 mmol) were added to the resulting solution. After
stirring for 16 h, volatiles were removed under reduced pressure. The residue was dissolved in
EtOAc, and the resulting solution was washed successively with 0.10 M KHSO4 (2×) and
saturated aqueous NaHCO3 (2×). The organic layer was dried over Na2SO4(s), filtered, and
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concentrated under reduced pressure. The residue was dissolved in the minimum volume of
DCM and triturated with hexanes. To enhance its solidity, material insoluble in hexanes was
dissolved in methanol, and volatiles were removed under reduced pressure to give FmocꢀDꢀProꢀ
DꢀProꢀGlyꢀOtBu (5.26 g, 82%) as an offꢀwhite solid, which was judged to be 95% pure by
LCMS. HRMSꢀESI (m/z): [M + H]+ calcd, 548.2756; found, 548.2755. 1H NMR (400 MHz,
CDCl3, δ): 7.85–7.27 (m, 8H), 4.81–4.13 (m, 2H), 4.05–3.36 (m, 6H), 3.34–2.66 (m, 2H), 2.36–
1.62 (m, 9H), 1.56–1.37 (m, 9H). 13C NMR (101 MHz, CDCl3, δ): 171.32, 168.87, 140.13,
138.01, 134.70, 129.09, 128.73, 127.05, 124.34, 121.00, 120.32, 119.74, 107.79, 82.10, 77.36,
77.24, 77.04, 76.72, 59.96, 59.24, 47.68, 46.79, 42.04, 41.98, 30.49, 28.04, 27.38, 26.28, 25.01.
Synthesis of Fmoc-D-Pro-D-Pro-Gly-OH
FmocꢀDꢀProꢀDꢀProꢀGlyꢀOtBu (5.26 g, 9.6 mmol) was dissolved in 12 mL of 4 M HCl in dioxane,
and the resulting solution was stirred for 14 h at room temperature. The reaction mixture was
concentrated under reduced pressure. The residue was dissolved in the minimum volume of
DCM and triturated with hexanes. Material insoluble in hexanes was dissolved in methanol, and
volatiles were removed under reduced pressure to give FmocꢀDꢀProꢀDꢀProꢀGlyꢀOH (3.37 g, 71%
yield) as an offꢀwhite solid, which was judged to be 90% pure by LCMS. HRMSꢀESI (m/z): [M
+ H]+ calcd, 492.2130; found, 492.2133. 1H NMR (400 MHz, CDCl3, δ): 7.75 (t, J = 7.6 Hz,
2H), 7.57 (ddd, J = 25.7, 15.6, 7.3 Hz, 2H), 7.39 (td, J = 7.7, 3.1 Hz, 2H), 7.30 (tt, J = 7.5, 1.3
Hz, 2H), 4.73–4.48 (m, 1H), 4.47–4.18 (m, 3H), 4.18–3.80 (m, 2H), 3.80–3.63 (m, 2H), 3.63–
3.48 (m, 2H), 2.61–1.65 (m, 8H).13C NMR (101 MHz, CDCl3, δ): 172.34, 172.19, 172.15,
172.12, 171.91, 171.85, 171.82, 171.68, 155.43, 155.12, 154.45, 144.31, 144.11, 144.01, 143.78,
143.64, 141.28, 141.24, 141.22, 127.77, 127.72, 127.69, 127.59, 127.14, 127.10, 127.07, 127.01,
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125.26, 125.12, 124.81, 124.65, 119.96, 119.77, 67.85, 67.63, 66.28, 60.94, 60.00, 59.67, 58.73,
58.40, 57.67, 47.58, 47.24, 47.16, 47.05, 46.88, 46.64, 41.42, 41.34, 41.27, 31.84, 30.14, 29.33,
29.28, 27.87, 27.66, 25.12, 24.99, 24.64, 24.40, 23.09, 22.03, 20.79.
Synthesis of Boc-Pro-Pro-OMe
BocꢀProꢀOH (3.97 g, 18 mmol) and NMM (6.0 mL, 55 mmol) were dissolved in 200 mL of
THF, and the resulting solution was cooled to –78 °C in a dry ice/acetone bath. Isobutyl
chloroformate (2.4 mL, 18 mmol) was added dropwise, and a white precipitate (NMM·HCl)
formed. Solid HClꢁProꢀOMe (3.05 g, 18 mmol) was added in one portion, and the reaction
mixture was allowed to warm to room temperature and then stirred for 12 h. The precipitate was
removed by filtration, and the solution was concentrated under reduced pressure. The residue
was dissolved in EtOAc, and the resulting solution was washed successively with 0.10 M
KHSO4 (2×) and saturated aqueous NaHCO3 (2×). The organic layer was dried over Na2SO4(s),
filtered, and volatiles were removed under reduced pressure to give BocꢀProꢀProꢀOMe (3.25 g,
54%) as a pale yellow solid. Also present is the byproduct of the carbamate formed by reaction
of HClꢁProꢀOMe and isobutyl chloroformate. HRMSꢀESI (m/z): [M + H]+ calcd, 327.1915;
found, 327.1912. 1H NMR (400 MHz, CDCl3, δ): 4.57 (ddd, J = 13.3, 8.5, 4.2 Hz, 0H), 4.50 (dd,
J = 8.4, 3.1 Hz, 0H), 4.38 (td, J = 8.6, 3.9 Hz, 0H), 4.32 (dd, J = 8.7, 3.8 Hz, 0H), 3.95–3.85 (m,
1H), 3.82–3.75 (m, 0H), 3.75–3.69 (m, 3H), 3.66–3.32 (m, 3H), 2.32–1.74 (m, 7H), 1.45 (s, 3H),
1.39 (s, 2H), 0.94 (d, J = 6.7 Hz, 1H), 0.89 (d, J = 6.7 Hz, 2H). 13C NMR (101 MHz, CDCl3, δ):
173.42, 173.26, 172.92, 172.63, 171.60, 171.12, 154.62, 154.57, 153.71, 79.44, 71.45, 59.01,
58.73, 58.67, 57.73, 57.68, 52.18, 52.08, 46.84, 46.77, 46.64, 46.46, 46.25, 30.94, 29.98, 29.90,
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29.05, 28.81, 28.70, 28.49, 28.34, 28.02, 27.96, 25.03, 24.97, 24.31, 24.05, 23.55, 19.04, 18.92,
18.89.
Synthesis of Fmoc-Gly-Pro-Pro-OMe
BocꢀProꢀProꢀOMe (3.25 g, 9.9 mmol) was dissolved in 9.0 mL of 4 M HCl in dioxane. The
resulting solution was stirred for 14 h at room temperature. The reaction mixture was
concentrated under reduced pressure. The residue was dissolved in 30 mL of THF, and NMM
(3.28 mL, 29 mmol) and FmocꢀGlyꢀOPfp (4.56 g, 9.9 mmol) were added to the resulting
solution. After stirring for 16 h, the reaction mixture was concentrated under reduced pressure.
The residue was dissolved in EtOAc, and the resulting solution was washed successively with
0.10 M KHSO4 (2×), saturated aqueous NaHCO3 (2×). The organic layer was dried over
Na2SO4(s), filtered, and concentrated under reduced pressure. The residue was dissolved in the
minimum volume of DCM and triturated with hexanes. To enhance its solidity, material
insoluble in hexanes was dissolved in methanol, and volatiles were removed under reduced
pressure to give FmocꢀGlyꢀProꢀProꢀOMe (4.38 g, 87%) as an offꢀwhite solid, which was judged
to be 90% pure by LCMS. HRMSꢀESI (m/z): [M + H]+ calcd, 506.2292; found, 506.2286. 1H
NMR (400 MHz, CDCl3, δ): 7.76 (dd, J = 7.6, 4.9 Hz, 2H), 7.59 (dd, J = 7.6, 3.6 Hz, 2H), 7.43–
7.35 (m, 2H), 7.30 (tdd, J = 7.6, 6.0, 1.5 Hz, 2H), 5.73 (t, J = 4.4 Hz, 1H), 5.42 (d, J = 23.5 Hz,
1H), 4.70 (dd, J = 8.0, 3.7 Hz, 1H), 4.57 (dd, J = 8.6, 4.2 Hz, 1H), 4.41 (d, J = 7.2 Hz, 1H), 4.36
(dd, J = 7.3, 3.3 Hz, 1H), 4.22 (q, J = 7.7 Hz, 1H), 4.13 (dd, J = 17.2, 5.7 Hz, 1H), 4.05 (s, 1H),
4.03–3.91 (m, 1H), 3.83 (dt, J = 9.5, 7.1 Hz, 1H), 3.77 (s, 1H), 3.72 (s, 2H), 3.63 (dt, J = 9.7, 6.2
Hz, 2H), 3.57–3.43 (m, 1H), 2.22 (qd, J = 11.5, 10.3, 7.1 Hz, 2H), 2.13–1.93 (m, 4H). 13C NMR
(101 MHz, CDCl3, δ): 172.57, 170.51, 167.21, 156.38, 143.89, 143.84, 141.29, 141.25, 127.74,
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127.67, 127.09, 127.07, 127.05, 125.19, 125.08, 120.00, 119.97, 119.93, 67.24, 63.81, 58.90,
58.22, 53.58, 52.43, 52.28, 47.09, 46.79, 46.36, 43.34, 28.79, 28.20, 24.93, 24.61.
Synthesis of Fmoc-Gly-Pro-Pro-OH
FmocꢀGlyꢀProꢀProꢀOMe (4.38 g, 8.6 mmol) was dissolved in 50 mL of 80:20 isopropanol/THF.
Powdered CaCl2 (14.22 g, 128 mmol), LiOHꢁH2O (1.42 g, 33 mmol), and H2O (5 mL) were
added to the resulting solution. The mixture was vigorously stirred for 48 h. The pH was adjusted
to 2 with aqueous HCl (1% v/v), and the resulting solution was concentrated under reduced
pressure. The aqueous layer was extracted with EtOAc, and the organic layer was washed with
water and brine, dried over Na2SO4(s), and concentrated under reduced pressure. The residue
was dissolved in the minimum volume of DCM and triturated with hexanes. To enhance its
solidity, material insoluble in hexanes was dissolved in methanol, and volatiles were removed
under reduced pressure to give FmocꢀGlyꢀProꢀProꢀOH (3.63 g, 86%) as an offꢀwhite solid, which
was judged to be 87% pure by LCMS. HRMSꢀESI (m/z): [M + H]+ calcd, 492.2130; found,
492.2128. 1H NMR (400 MHz, CDCl3, δ): 7.75 (d, J = 7.5 Hz, 2H), 7.64–7.54 (m, 2H), 7.39 (t, J
= 7.5 Hz, 2H), 7.33–7.27 (m, 2H), 5.77 (s, 1H), 5.39 (s, 1H), 4.82–4.51 (m, 2H), 4.49–4.29 (m,
2H), 4.21 (t, J = 7.3 Hz, 1H), 4.14 (dd, J = 17.3, 5.8 Hz, 1H), 4.06–3.88 (m, 1H), 3.83 (dd, J =
9.4, 7.2 Hz, 1H), 3.74–3.45 (m, 3H), 2.43–1.79 (m, 6H). 13C NMR (101 MHz, CDCl3, δ):
174.38, 172.80, 171.53, 167.89, 156.72, 156.68, 143.88, 143.79, 143.74, 141.25, 127.74, 127.70,
127.09, 127.06, 125.20, 125.17, 125.10, 119.98, 119.95, 67.31, 59.31, 58.99, 58.26, 52.35, 47.13,
47.07, 47.03, 46.47, 43.27, 42.58, 28.77, 28.39, 28.19, 24.92, 24.88, 24.70.
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Synthesis of Cbz-Hyp(tBu)-Gly-OMe
CbzꢀHyp(tBu)ꢀOH (3.01 g, 9.4 mmol) and Nꢀmethyl morpholine (NMM) (3.1 mL, 28 mmol)
were dissolved in 200 mL of THF, and the resulting solution was cooled to –78 °C in a dry
ice/acetone bath. Isobutyl chloroformate (1.2 mL, 9.3 mmol) was added dropwise, and a white
precipitate (NMM·HCl) formed. Solid HClꢁGlyOMe (1.17 g, 9.3 mmol) was added in one
portion, and the reaction mixture was allowed to warm to room temperature and then stirred for
12 h. The precipitate was removed by filtration, and the solvent was concentrated under reduced
pressure. The residue was dissolved in EtOAc, and the resulting solution was washed
successively with 0.10 M KHSO4 (2×) and saturated aqueous NaHCO3 (2×). The organic layer
was dried over Na2SO4(s), filtered, and volatiles were removed under reduced pressure to give
CbzꢀHyp(tBu)ꢀGlyꢀOMe (3.26 g, 88%) as a pale yellow solid, which was judged to be 99% pure
by LCMS. HRMSꢀESI (m/z): [M + H]+ calcd, 393.2021; found, 393.2015. Integration of signals
for the N–H proton revealed that the cis:trans ratio of the prolyl peptide bond was 1.0:2.1. 1H
NMR (400 MHz, CDCl3, δ): 7.39–7.28 (m, 3H), 7.20 (s, 1H), 6.32 (s, 1H), 5.34–4.93 (m, 2H),
4.58–4.20 (m, 2H), 4.03 (d, J = 5.4 Hz, 1H), 3.74 (s, 3H), 3.64 (dd, J = 10.5, 7.2 Hz, 1H), 3.26
(dd, J = 10.6, 6.6 Hz, 1H), 2.53–2.35 (m, 1H), 2.19 (d, J = 22.4 Hz, 1H), 1.94 (q, J = 10.5, 9.7
Hz, 1H), 1.72 (s, 1H), 1.18 (s, 9H). 13C NMR (100 MHz, CDCl3, δ): 171.76, 170.06, 156.26,
136.30, 128.52, 128.12, 127.84, 69.48, 67.45, 58.95, 52.96, 52.34, 41.24, 35.99, 28.26.
Synthesis of Fmoc-Pro-Hyp(tBu)-Gly-OMe
A suspension of CbzꢀHyp(tBu)ꢀGlyꢀOMe (3.26 g, 8.3 mmol) and Pd/C (877 mg, 10% w/w) in
25 mL of MeOH was stirred under an atmosphere of H2(g) for 16 h at room temperature. Any
insoluble material was removed by filtration, and volatiles were removed under reduced
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pressure. (The weight of the crude intermediate was 2.0 g.) The residue was dissolved in 35 mL
of THF, and NMM (2.4 mL, 22 mmol) and FmocꢀProꢀOPfp (2.91 g, 5.8 mmol) were added to
the resulting solution. After stirring for 16 h, the reaction mixture was concentrated under
reduced pressure. The residue was dissolved in EtOAc, and the resulting solution was washed
successively with 0.10 M KHSO4 (2×) and saturated aqueous NaHCO3 (2×). The organic layer
was dried over Na2SO4(s), filtered, and concentrated under reduced pressure. The residue was
dissolved in the minimum volume of DCM and triturated with hexanes. To enhance its solidity,
material insoluble in hexanes was dissolved in methanol, and volatiles were removed under
reduced pressure to give FmocꢀProꢀHyp(tBu)ꢀGlyꢀOMe (1.23 g, 25%) as an offꢀwhite solid,
which was judged to be 96% pure by LCMS. HRMSꢀESI (m/z): [M + H]+ calcd, 578.2861;
found, 578.2866. 1H NMR (500 MHz, CDCl3, δ): 7.77 (dd, J = 7.5, 3.9 Hz, 2H), 7.68–7.52 (m,
2H), 7.40 (t, J = 7.7 Hz, 2H), 7.36–7.29 (m, 2H), 4.64–4.19 (m, 6H), 4.10 (ddd, J = 23.8, 18.0,
6.6 Hz, 1H), 4.00–3.79 (m, 1H), 3.79–3.43 (m, 6H), 2.72–2.39 (m, 1H), 2.37–1.83 (m, 6H),
1.44–1.01 (m, 9H). 13C NMR (126 MHz, CDCl3, δ): 172.93, 171.86, 170.40, 141.65, 128.04,
127.44, 125.63, 125.49, 120.40, 120.34, 77.16, 74.48, 70.57, 67.92, 58.78, 52.63, 47.61, 47.21,
41.42, 34.82, 29.66, 28.67, 28.62, 25.15, 24.75.
Synthesis of Fmoc-Pro-Hyp(tBu)-Gly-OH
FmocꢀProꢀHyp(tBu)ꢀOMe (1.23 g, 2.1 mmol) was dissolved in 30 mL of 80:20
isopropanol/THF. Powdered CaCl2 (3.53 g, 32 mmol), LiOH·H2O (355 mg, 8.5 mmol), and
water (5 mL) were added to the resulting solution. The mixture was stirred vigorously for 12 h.
The pH was adjusted to 2 with 1% w/v aqueous HCl, and the resulting solution was concentrated
under reduced pressure. The aqueous layer was extracted with EtOAc, and the organic layer was
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washed with water and brine, dried over Na2SO4(s), and concentrated under reduced pressure.
The residue was dissolved in the minimum volume of DCM and triturated with hexanes.
Material insoluble in hexanes was dissolved in methanol, and volatiles were removed under
reduced pressure to give FmocꢀProꢀHyp(tBu)ꢀGlyꢀOH (978 mg, 83% yield) as an offꢀwhite solid,
which was judged to be 94% pure by LCMS. 1H NMR (500 MHz, CDCl3, δ): 7.85–7.76 (m, 2H),
7.71–7.54 (m, 2H), 7.44 (td, J = 7.4, 3.0 Hz, 2H), 7.35 (ddt, J = 8.6, 7.4, 1.2 Hz, 2H), 4.91–3.45
(m, 12H), 2.78–1.59 (m, 7H), 1.50–0.92 (m, 9H).13C NMR (126 MHz, CDCl3, δ): 172.27,
172.21, 172.09, 172.04, 172.00, 171.96, 171.65, 155.50, 155.19, 154.63, 144.18, 144.10, 144.00,
143.73, 143.57, 141.29, 141.26, 141.23, 141.14, 127.78, 127.71, 127.68, 127.13, 127.09, 127.05,
125.23, 125.08, 124.76, 124.72, 120.03, 119.99, 119.94, 74.50, 74.27, 74.18, 70.15, 69.91, 67.92,
67.69, 67.37, 67.10, 59.48, 58.86, 58.64, 58.44, 58.04, 57.78, 53.38, 52.74, 47.32, 47.15, 47.02,
46.87, 41.37, 41.25, 35.62, 35.35, 30.14, 29.12, 28.22, 28.20, 28.16, 24.62, 24.33, 23.02.
Synthesis of Ac-Lys-(Ser-Gly)3-(D-Pro-D-Pro-Gly)7
Using the FmocꢀDꢀProꢀDꢀProꢀGlyꢀOH tripeptide synthesized in solution without chromatography
(vide supra) and an FmocꢀDꢀProꢀOH monomer, AcꢀLysꢀ(SerꢀGly)3ꢀ(DꢀProꢀDꢀProꢀGly)7 was
synthesized by two additions of monomer followed by six segment condensations of tripeptide
on preloaded FmocꢀGlyꢀ2ꢀchlorotrityl resin (0.19 mmol/g) from EMD Millipore (La Jolla, CA).
Fmocꢀdeprotection was achieved by treatment with piperidine (20% v/v) in DMF. The tripeptide
or amino acid monomer (4 equiv) was converted to an active ester using HATU and NMM. Each
residue was doubleꢀcoupled between Fmocꢀdeprotections.
Peptide was cleaved from the resin with 96.5:2.5:1.0 TFA/H2O/TIPSH (5 mL), precipitated
from diethyl ether at 0 °C, and isolated by centrifugation. The peptide was purified by
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preparative HPLC using a gradient of 10–50% v/v B over 50 min (A: H2O containing 0.1% v/v
TFA; B: acetonitrile containing 0.1% v/v TFA). MALDI (m/z): [M + H]+ calcd 2380.6, found
2380.0. A 50ꢀµmol scale synthesis afforded 18.2 mg (15%) of AcꢀLysꢀ(SerꢀGly)3ꢀ(DꢀProꢀDꢀProꢀ
Gly)7 after purification.
RESULTS AND DISCUSSION
The key to establishing a route for chromatographyꢀfree peptide synthesis is to mitigate
byproduct formation. To do so, we coupled amino acids by using isobutyl chloroformate45 to
form transient anhydrides or by using stable pentafluorophenyl esters.44 These known reagents46
produce volatile and waterꢀsoluble byproducts, respectively. We applied variations of our
method to four tripeptides that are used commonly in CMP strands (Scheme 1). These tripeptides
are rich in glycine (which lacks a stereogenic center) and proline (which has virtually no
tendency to epimerize during coupling reactions).47ꢀ49 FmocꢀProꢀProꢀGlyꢀOH and its
permutation, FmocꢀGlyꢀProꢀProꢀOH, provide the simplest triplets in CMPs. FmocꢀDꢀProꢀDꢀProꢀ
GlyꢀOH is an enantiomer that can serve as a control for experiments in chiral environments.
Finally, FmocꢀProꢀHyp(tBu)ꢀGlyꢀOH provides the most common triplet found in natural
collagen, and contains an acidꢀlabile sideꢀchain protecting group.27 We were able to access each
of these tripeptides without the use of chromatography (Table 1).
As an example, we elaborate on the synthesis of FmocꢀProꢀProꢀGlyꢀOH (Scheme 2). The
first coupling step was accomplished by activating the carboxylic acid with isobutyl
chloroformate and NMM. Isobutyl chloroformate is a relatively inexpensive reagent compared to
many common coupling reagents (e.g., HATU). Coupling with isobutyl choroformate enables
ready access to a relatively pure product, as the two major byproducts—CO2 and isobutanol—are
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volatile.50 Insoluble NMM·HCl is removed by filtration, and any residual NMM (bp 115 °C) is
removed by evaporation or by aqueous extraction along with unreacted starting material (e.g., the
free amine and free acid, which are nonꢀvolatile). This route provided CbzꢀProꢀGlyꢀOtBu in 92%
yield.
Deprotection of the amino group of the dipeptide by hydrogenolysis was followed
immediately by addition of the third amino acid, FmocꢀProꢀOPfp. Again, the byproduct,
pentafluorophenol is soluble in the aqueous layer during an extraction. Pentafluorophenol
(bp 143 °C) also has modest volatility. Initially, we attempted this coupling reaction with
N,Nꢀdiisopropylethylamine as the base in DMF. These conditions provided the desired tripeptide,
but LCMS showed evidence of some proline oligomers, presumably from inadvertent Fmoc
deprotection. Changing to a weaker base—NMM (which has a conjugate acid of pKa 7.4151)—in
THF eliminated this problem and afforded FmocꢀProꢀProꢀGlyꢀOtBu in 74% yield.
Finally, the tꢀbutyl ester of FmocꢀProꢀProꢀGlyꢀOtBu was removed with anhydrous HCl in
dioxane, providing FmocꢀProꢀProꢀGlyꢀOH in 90% yield. In the analogous step en route to other
tripeptides, selective base saponification of a methyl ester in a wet organic solvent containing
LiOH (4 equiv) and CaCl2 (15 equiv) afforded the desired carboxylic acid without removing the
Fmoc group,52 affording FmocꢀGlyꢀProꢀProꢀOH and FmocꢀProꢀHyp(tBu)ꢀGlyꢀOH in 86% and
83% yield, respectively.
In lieu of chromatography, we used evaporation, extraction, and trituration to purify
products. In the first coupling step, isobutyl chloroformate was the coupling agent, and
evaporation and extraction were sufficient to provide a clean product. The second coupling and
final deprotection steps also employed trituration with hexanes, which does not dissolve the
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tripeptide product. The analytical liquid chromatography traces for each step in the synthesis of
FmocꢀProꢀProꢀGlyꢀOH exemplify the efficacy of these simple purification steps (Figure 1).
Like FmocꢀProꢀProꢀGlyꢀOH, three other tripeptides were obtained through this
chromatographyꢀfree methodology (Table 1). In general, coupling reactions proceeded in high
yield and provided product of high purity. We did note with 1H NMR spectroscopy that the
reaction of HClꢁProꢀOMe and isobutyl chloroformate generated a carbamate byproduct, which
was not observed in any other coupling. The carbamate impurity was, however, not detrimental
to subsequent steps and was readily removable by trituration, as shown by LCMS analysis and
by NMR spectroscopy.
The reaction of Hyp(tBu)ꢀGlyꢀOMe with FmocꢀProꢀOPfp was atypical in providing a low
(<50%) yield of product. We speculate that this deviation derives from particular steric and
electronic attributes of Hyp(tBu)ꢀGlyꢀOMe (Figure 2). Its tꢀbutyl protecting group is bulky and
could interfere with amide bond formation. Moreover, the sideꢀchain oxygen of Hyp is electronꢀ
withdrawing, thereby lowering the nucleophilicity of its amino group. Coupling to Hyp(tBu) was
likewise the lowest yielding step in a reported synthesis of FmocꢀProꢀHypꢀGlyꢀOH that was
chromatographyꢀfree but used crystallization to purify most reactions products.38 Yields might be
improved by using an unprotected Hyp monomer.38 Despite this one deviance, all other reactions
with the secondary amine of proline were highly successful. Moreover, each tripeptide product
was of suitable purity for SPPS.
In our work we have focused on the synthesis of amino acid trimers for CMPs. Nonetheless,
we envision that the methodology herein could be applicable to other peptides as well. In this
regard, the chromatographyꢀfree synthesis of FmocꢀProꢀHyp(tBu)ꢀGlyꢀOH, which demonstrates
tolerance for acidꢀlabile protecting groups, is especially noteworthy. We note as well that the
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pentafluorophenyl ester of FmocꢀDꢀPro is not available from commercial vendors. Accordingly,
we used HBTU as the coupling agent in the chromatographyꢀfree synthesis of FmocꢀDꢀProꢀDꢀ
ProꢀGlyꢀOH. These instances serve to showcase the generality of our procedures. Notably, our
route avoids hydrogenation of a molecule containing an Fmoc protecting group, which is
vulnerable to reduction.
Finally, we demonstrated the segment condensation of a tripeptide synthesized in solution
without chromatography. Specifically, we coupled FmocꢀDꢀProꢀDꢀProꢀGlyꢀOH units to effect the
solidꢀphase synthesis of AcꢀLysꢀ(SerꢀGly)3ꢀ(DꢀProꢀDꢀProꢀGly)7ꢀOH on a solid support. A mass
spectrum of the purified 28ꢀmer verified its integrity (Figure S2 of the Supporting Information).
CONCLUSION
We report on the synthesis of short peptides for use in SPPS by a novel combination of known
reagents and reaction conditions. The methodology enables the isolation of products by using the
facile techniques of evaporation, extraction, and trituration (Figure 3). Eliminating the need for
purification by column chromatography reduces waste, reduces cost, and increases the ease of
synthesis. This methodology is especially well suited for the synthesis of CMPs, which are in
widespread use.26,23,27,29,24,28,25,30ꢀ35
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Table 1. Yield and purity of intermediates and tripeptide products made by chromatography-free synthesis
Protected Dipeptide Ester
Cbz-Pro-Gly-OtBu
Yield
92%
97%
54%
88%
Puritya
99%
91%
ND
Fmoc Tripeptide Ester
Fmoc-Pro-Pro-Gly-OtBu
Fmoc-D-Pro-D-Pro-Gly-OtBu
Fmoc-Gly-Pro-Pro-OMe
Fmoc-Pro-Hyp(tBu)-Gly-OMe
Yield
74%
82%
87%
25%
Puritya
94%
Fmoc Tripeptide
Yield
Puritya
85%
Fmoc-Pro-Pro-Gly-OH
Fmoc-D-Pro-D-Pro-Gly-OH
Fmoc-Gly-Pro-Pro-OH
Fmoc-Pro-Hyp(tBu)-Gly-OH
90%
71%
86%
83%
Cbz-D-Pro-Gly-OtBu
Boc-Pro-Pro-OMe
95%
90%
90%
87%
Cbz-Hyp(tBu)-Gly-OMe
99%
96%
94%
aPurity was assessed with LCMS monitored at 254 nm. LCMS traces are shown in Figure 1 and the Supporting
Information. The absorbance of Boc-Pro-Pro-OMe at 254 nm was not detectable (ND).
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Scheme Legends
SCHEME 1 Four Fmocꢀprotected tripeptides synthesized by chromatographyꢀfree
methodology and their dipeptide precursors. Restrictions on utility are in italics typeface.
SCHEME 2 Synthetic route to FmocꢀProꢀProꢀGlyꢀOH that requires no chromatography.
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SCHEME 1
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SCHEME 2
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Figure Legends
FIGURE 1 LCMS traces of products from each step in the chromatographyꢀfree synthesis of
FmocꢀProꢀProꢀGlyꢀOH by the route in Scheme 2. Molecules were detected by asborance at
254 nm during LCMS with a reverseꢀphase column. Traces for the syntheses of other tripeptides
are shown in Figure S1 of the Supporting Information.
FIGURE 2 Steric and electronic effects that could diminish the reactivity of the amino group of
Hyp(tBu)ꢀGlyꢀOMe.
FIGURE 3 Summary of the physical properties of the products and byproducts from the
reactions herein.
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FIGURE 1
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FIGURE 2
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