Synthesis of fmoc-pro-hyp(TBDPS)-gly-OH and its application as a versatile building block for the preparation of collagen model peptides

Article abstract of DOI:10.1055/s-0028-1083281

The efficient synthesis of the tripeptidic building block Fmoc-Pro-Hyp(TBDPS)-Gly-OH and its application for the preparation of collagen model peptides (CMPs) has been achieved. The silyl ether protecting group prevents undesired side reactions during the

Full text of DOI:10.1055/s-0028-1083281

PAPER
143
Synthesis of Fmoc-Pro-Hyp(TBDPS)-Gly-OH and Its Application as a
Versatile Building Block for the Preparation of Collagen Model Peptides
Efficient
S
ynthes
o
is of Collage
m
n
M
odel Peptides an S. Erdmann, Helma Wennemers*
Department of Chemistry, University of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland
Fax +41(61)2670976; E-mail: Helma.Wennemers@unibas.ch
Received 20 October 2008
block are necessary. As a result, fewer side products form,
thereby facilitating the purification of the desired CMP.
Abstract: The efficient synthesis of the tripeptidic building block
Fmoc-Pro-Hyp(TBDPS)-Gly-OH and its application for the prepa-
ration of collagen model peptides (CMPs) has been achieved. The
silyl ether protecting group prevents undesired side reactions during
the CMP synthesis thereby facilitating purification and allowing for
selective deprotection of the hydroxyproline residue without affect-
ing the solid-supported CMP.
Especially the trimer strategy uses Hyp with an unprotect-
ed hydroxy function. Thus, in particular with potent cou-
pling reagents such as O-(6-chlorobenzotriazol-1-yl)-
N,N,N¢,N¢-tetramethyluronium
hexafluorophosphate
(HCTU), undesired side reactions involving the hydroxy
group can take place.8 Undesired acylation reactions can
also occur during capping with acetic anhydride (Ac2O)
after each coupling step; a protocol typically used to facil-
itate purification of the final product.8 An obvious solu-
tion to this problem is the protection of the hydroxy group
of Hyp. So far, tert-butyl ethers in particular have been
employed to protect Hyp during the SPPS synthesis of
CMPs.4b,7a This protecting group is readily removed under
the acidic conditions used for the cleavage of CMPs from
typically used solid supports such as Rink amide and
Wang resin. We sought an alternative protecting group
that could be selectively removed without simultaneous
cleavage of the CMP from these acid-sensitive solid sup-
ports. Here we present the synthesis of the tert-butyl-
diphenylsilyl (TBDPS) protected trimeric building block
Fmoc-Pro-Hyp(TBDPS)-Gly-OH and describe its appli-
cation in the solid-phase synthesis of a CMP consisting of
21 residues.
Key words: collagen, peptides, protecting groups, solid-phase syn-
thesis, TBDPS silyl ether
Collagen is the most abundant protein in vertebrates and
has applications in medicine and cosmetics.1 Collagen fi-
bers are assemblies of triple helices that consist of three
single strands with the repeat unit Xaa-Yaa-Gly where the
amino acids proline (Pro) and (4R)-hydroxyproline (Hyp)
are most common in the Xaa and Yaa positions, respec-
tively. Defects in the structure of collagen are involved in
several diseases such as scurvy and brittle bone disease.1–3
Thus, understanding the factors that influence the struc-
ture of collagen is important. Towards this goal, various
collagen model peptides (CMPs) comprising typically
21–30 residues have been prepared in order to investigate
their structural properties.3–6 Since the Pro-Hyp-Gly motif
is the most common natural repeat unit within collagen, it
is also the most common motif used within CMPs.3–6
Thus, the effective synthesis of CMPs with at least seven
repeat units of Pro-Hyp-Gly is an important goal.
Aside from the requirement of allowing for selective re-
moval from resin-bound CMPs under mild conditions, a
suitable protecting group must also allow for facile intro-
duction and has to be stable during the SPPS and the syn-
thesis of the trimeric Fmoc-protected building block
Fmoc-Pro-Hyp(PG)-Gly-OH. In addition, a sterically de-
manding protecting group is desirable in order to prevent
aggregation of the peptide during SPPS. Silyl ethers with
aryl and/or sterically demanding alkyl substituents are
versatile protecting groups for hydroxy groups that can be
easily introduced, and can be removed either with fluoride
ions or under acidic conditions but are otherwise robust.
We therefore envisioned a silyl ether to be a suitable pro-
tecting group for our improved synthesis of CMPs
(Figure 1).
Two different strategies for the synthesis of CMPs are
used. Either single Fmoc-protected amino acids are cou-
pled using solid-phase peptide synthesis (SPPS), or tri-
meric building blocks such as Fmoc-Pro-Hyp-Gly-OH are
prepared in solution phase and then utilized in SPPS.5–7
The first method relies on large excesses of the Fmoc-pro-
tected amino acid building blocks and the coupling re-
agents (5–10 equiv) in every coupling step. This approach
can lead to the formation of significant amounts of trun-
cated fragments in which the trimeric repeat unit is not
preserved. These impurities can disrupt triple helix forma-
tion and are often difficult to separate from the desired
CMP. The second method requires the preparation of the
trimeric building block (e.g., Fmoc-Pro-Hyp-Gly-OH) in
solution phase.7 This approach is advantageous since few-
er coupling steps with less equivalents of the building
R1
R2
Si
O
R3
O
H
N
N
N
Fmoc
OH
SYNTHESIS 2009, No. 1, pp 0143–0147
0
2
.
0
1
.2
0
0
9
Advanced online publication: 12.12.2008
O
O
DOI: 10.1055/s-0028-1083281; Art ID: C05308SS
© Georg Thieme Verlag Stuttgart · New York
Figure 1 Silyl ether protected building block for CMP synthesis

144
HCl⋅HN
b
R. S. Erdmann, H. Wennemers
PAPER
the diketopiperazine consisting of Pro-Hyp(TBDPS) were
observed as a byproduct. Fmoc-protection of the amino
group with Fmoc-OSu under Schotten–Baumann condi-
tions provided the trimeric building block Fmoc-Pro-
Hyp(TBDPS)-Gly-OH (4) in 86% yield over two steps.
Thus, the target building block 4, ready for SPPS was ob-
tained in an overall yield of 68% (Scheme 1).
OH
OH
a
OH
N
N
OH
Cbz
O
O
O
1
OR
O
In order to evaluate building block 4 in the SPPS of
CMPs, seven successive coupling and Fmoc-deprotection
steps were performed on Rink amide resin with a loading
of 0.36 mmol/g. HCTU/i-Pr2NEt served as coupling re-
agent, and a solution of piperidine in DMF (1:4) was used
for Fmoc deprotection (Scheme 2). After each coupling,
acetic anhydride was used to cap any remaining free ami-
no groups. The protocol proved efficient enough to be per-
formed in an automated fashion on a peptide synthesizer
for the assembly of CMP 5 on Rink amide resin. For the
cleavage of the silyl ethers from the Hyp-residues, resin-
bound CMP 5 was agitated in a 1 M solution of tetrabutyl
ammonium fluoride (TBAF) in tetrahydrofuran over-
night. The resin was then washed with tetrahydrofuran
and dichloromethane before removing CMP 6 from the
solid phase using a solution of trifluoroacetic acid (TFA)
in dichloromethane.
H
N
N
N
Cbz
OBn
O
O
2 R = H
c
3 R = TBDPS
OTBDPS
d
O
H
N
N
N
Fmoc
OH
O
O
4
Scheme 1 Reagents and conditions: (a) (i) Benzyltrimethylammo-
nium hydroxide, MeOH, r.t.; (ii) Cbz-Pro-OSu, DMF, r.t., 89%; (b)
H-Gly-OH·HCl, EDC·HCl, HOBt, i-Pr2NEt, DMF, r.t., 95%; (c)
TBDPS-Cl, imidazole, DMF, r.t., 93%; (d) (i) HCO2NH4, Pd/C, Me-
OH, r.t.; (ii) Fmoc-OSu, Et3N, MeCN–H2O, r.t., 86%.
Benzyl groups were anticipated to be the most suitable
protecting groups for the amino and carboxylic acid func-
tionalities during the synthesis of the trimeric Fmoc-pro-
tected building block Fmoc-Pro-Hyp(PG)-Gly-OH. We
therefore started our synthesis by condensation of the
commercially available Cbz-protected activated succin-
imide ester of proline (Cbz-Pro-OSu) with Hyp under pre-
viously described conditions to isolate the dipeptide Cbz-
Pro-Hyp-OH (1) in 89% yield (Scheme 1).7a Dipeptide 1
was coupled with the glycine benzyl ester H-Gly-OBn us-
ing EDC/HOBt as coupling reagents to provide tripeptide
Cbz-Pro-Hyp-Gly-OBn (2)7a in 95% yield. We then
investigated the utility of the tert-butyldimethylsilyl
(TBDMS) ether as a protecting group. Introduction using
TBDMS-Cl in the presence of imidazole proceeded
smoothly, however, upon hydrogenation of the benzyl
protecting groups using Pd/C under a hydrogen atmo-
sphere, cleavage of the TBDMS ether was observed. Sev-
eral different conditions were tested, however, either
cleavage of the TBDMS ether or incomplete removal of
the benzyl protecting groups was observed.9 We therefore
turned our attention to the sterically more demanding and
stable tert-butyldiphenylsilyl (TBDPS) protecting group.
Protection of the Hyp residue within tripeptide 2 using
TBDPS-Cl and imidazole proceeded smoothly (93%
yield) to provide the protected peptide Cbz-Pro-
Hyp(TBDPS)-Gly-OBn (3). Hydrogenation with Pd/C
and H2 proved sluggish and incomplete cleavage of the
Cbz group was typically observed. In contrast, phase-
transfer hydrogenation with ammonium formate as the
hydrogen source and Pd/C as catalyst led to the C- and N-
terminally unprotected tripeptide silyl ether H-Pro-
Hyp(TBDPS)-Gly-OH within 45 minutes. Only traces of
FmocHN
Rink amide resin
a) piperidine–DMF (1:4)
b) 4 (4.5 equiv), HCTU (4.5 equiv), i-Pr2NEt (13.5 equiv)m
c) repeat steps a) and b) 6 times
OTBDPS
O
H
N
N
N
Fmoc
N
H
O
O
7
5
a) piperidine–DMF (1:4)
b) TBAF (1 M in THF)
c) TFA–CH2Cl2 (2:1)
OH
O
H
N
N
N
TFA⋅H
NH2
O
O
7
6
Scheme 2 Solid-phase synthesis of CMP 6
Analysis of CMP 6 by HPLC demonstrated a purity of
≥60% of the crude material, most of the other signals
observed in the chromatogram correspond to fragments
derived from incomplete couplings (Figure 2). For com-
parison, CMP 6 was also prepared under similar condi-
tions (without capping with Ac2O) using the trimeric
building block Fmoc-Pro-Hyp-Gly-OH without a protect-
ing group on the Hyp residue. The chromatogram of the
crude material derived from this synthesis indicated a pu-
Synthesis 2009, No. 1, 143–147 © Thieme Stuttgart · New York

PAPER
Efficient Synthesis of Collagen Model Peptides
145
Cbz-Pro-Hyp-OH (1)
rity of only ~35%. Purification of CMP 6 prepared by the
strategy described above by preparative HPLC proved
straightforward and provided 6 in a purity of ≥98% in a
yield ranging from 20–40% with respect to the original
loading of the resin used. In contrast, yields of the analo-
gous purified CMPs prepared without protecting groups
on Hyp ranged from 10 to 20%.
H-Hyp-OH (10.5 g, 79.7 mmol, 1.0 equiv) was suspended in ben-
zyltrimethlyammonium hydroxide in MeOH (40% w/w, 47.1 mL,
103 mmol, 1.3 equiv) and MeOH (10 mL) was added until a yellow
solution was obtained. The solution was stirred for 10 min then all
volatiles were removed under reduced pressure. The residual yellow
oil was dissolved in DMF (60 mL) and Cbz-Pro-OSu (25.0 g, 72.2
mmol, 0.91 equiv) was added. The yellow solution was stirred over-
night then all volatiles were removed under reduced pressure. The
yellow residue was dissolved in NaHCO3 (5%, 200 mL) and washed
with Et2O (3 × 150 mL). The aqueous layer was slowly added to 3
M HCl (200 mL) and treated in an ultrasonic bath until precipitation
of a white solid was observed. The solid was filtered, washed with
cold 1 M HCl (3 × 50 mL) and dried under high vacuum. The ana-
lytical data of 1 are identical to those reported by Moroder and co-
workers.7a
Yield: 23.4 g (89%); white solid; Rf = 0.51 (MeCN–CH2Cl2–AcOH,
8:1:1; KMnO4).
1H NMR (400 MHz, DMSO-d6): d = 12.84 (s, 1 H), 7.40–7.23 (m,
5 H), 5.11–4.93 (m, 2 H), 4.59–4.47 (m, 1 H), 4.40–4.32 (m, 1 H),
4.25 (q, J = 7.9 Hz, 1 H), 3.62–3.32 (m, 4 H), 2.29–1.99 (m, 2 H),
1.95–1.73 (m, 4 H).
13C NMR (100 MHz, DMSO-d6): d = 173.2 (COOH), 170.3, 170.0
(amide), 153.8, 153.5 (carbamate), 137.0, 128.3, 128.2, 127.7,
127.4, 126.7 (Cbz), 69.0, 68.9 (CgHyp), 65.7, 65.6 (Cbz), 58.0, 57.6,
57.4 (CaHyp, CaPro), 54.3, 54.2 (CdHyp), 46.9, 46.2 (CdPro), 36.9
(CbHyp), 29.3, 28.3 (CbPro), 23.5, 22.8 (CgPro).
ESI-MS: m/z calcd for C18H22N2O6: 362.2; found: 385.3 [M + Na]+
(100%), 747.3 [2M + Na]+ (30%).
Cbz-Pro-Hyp-Gly-OBn (2)
Dipeptide 1 (16.4 g, 45.3 mmol, 1.0 equiv) and H-Gly-OBn·HCl
(11.0 g, 54.4 mmol, 1.2 equiv) were suspended in DMF (140 mL)
and cooled to 0 °C. The suspension was stirred and HOBt (6.94 g,
45.3 mmol, 1.0 equiv) and i-Pr2NEt (7.31 mL, 47.6 mmol, 1.05
equiv) were added, which gave a clear solution. EDC·HCl (9.56 g,
49.9 mmol, 1.1 equiv) was added and the reaction mixture was
stirred overnight. The solvent was evaporated under reduced pres-
sure and the yellow residue was dissolved in CH2Cl2 (300 mL) and
washed with sat. NaHCO3 (3 × 150 mL). After washing with aq
KHSO4 (10%; 3 × 150 mL) the organic layer was dried over MgSO4
and all volatiles were removed under reduced pressure. Drying un-
der high vacuum gave 2. The analytical data of 2 are identical to
those reported by Moroder and co-workers.7a
Figure 2 RP-HPLC chromatograms of CMP 6 before (top) and
after (bottom) preparative HPLC purification
In conclusion, we have developed the silyl ether protected
tripeptide Fmoc-Pro-Hyp(TBDPS)-Gly-OH as a versatile
building block for the synthesis of CMPs. The TBDPS
protecting group prevents undesired acylation reactions
during the synthesis, thereby facilitating the purification
and increasing the yield of the CMPs. In addition, the
bulkiness of the TBDPS might prove helpful in preventing
aggregation of longer CMPs on the solid support.
Yield: 22.1 g (96%); white solid; Rf = 0.35 (MeOH–CH2Cl2, 5%;
KMnO4).
1H NMR (400 MHz, CDCl3): d = 7.60 (t, J = 5.6 Hz, 1 H, NHGly),
7.40–7.21 (m, 10 H, Cbz, BnOCO), 5.30–4.94 [m, 4 H,
PhCH2O(CO)N, PhCH2OCO], 4.73 (t, J = 7.7 Hz) and 4.66–4.27
(m, 3 H, HaPro, HaHyp, HgHyp), 4.10–3.30 (m, 6 H, HaGly, HdPro,
HdHyp), 2.47–1.67 (m, 6 H, HbPro, HbHyp, HgPro).
13C NMR (100 MHz, CDCl3): d = 172.0, 171.7, 169.5 (amides, es-
ter), 155.1 (carbamate), 136.4, 135.3, 128.5, 128.4, 128.3, 128.2,
127.9, 127.7 (Cbz, Bn), 70.4 (CgHyp), 67.1, 66.8 (Cbz, Bn), 58.4,
58.3 (CaPro, CaHyp), 55.0 (CdHyp), 46.8 (CdPro), 41.3 (CaGly),
36.4 (CbHyp), 29.4 (CbPro), 24.3 (CgPro).
Materials and reagents were of the highest commercially available
grade and used without further purification. Reactions were moni-
tored by TLC using Merck silica gel 60 F254 plates. Compounds
were visualized by UV, ninhydrin and KMnO4. Flash chromatogra-
phy was performed using Merck silica gel 60 (40–63 mm). 1H and
13C NMR spectra were recorded on Bruker DPX 500 and DPX 400
1
spectrometers. The H and 13C NMR spectra of compounds 1–4
contained at least two sets of peaks due to the cis and trans conform-
ers around the tertiary carbamate and amide bonds. Peak assign-
ments are not listed separately for the conformers. Chemical shifts
(d) are reported in ppm using TMS as a reference. Bruker Esquire
3000 Plus was used for electrospray ionization (ESI) mass spec-
trometry measurements. High-resolution mass spectra were record-
ed on a Sciex QStar Pulsar electrospray ionization time-of-flight
spectrometer (ESI-TOF). Analytical HPLC was performed using a
LiChrospher 100 RP-18e (5 mm, 250 mm × 4 mm) column from
Merck. Preparative HPLC was carried out on a LiChrospher RP-18e
(5 mm, 250 mm × 10 mm) column from Merck. For automated pep-
tide synthesis, a Syro I Peptide Synthesizer (MultiSynTech GmbH,
Witten, Germany) was employed.
ESI-MS: m/z calcd for C27H31N3O7: 509.2; found: 532.4 [M + Na]+
(100%).
Cbz-Pro-Hyp(TBDPS)-Gly-OBn (3)
Tripeptide 2 (22.1 g, 43.3 mmol, 1.0 equiv) and imidazole (13.0 g,
191 mmol, 4.4 equiv) were dissolved in DMF (50 mL) and
TBDPS-Cl (31.0 mL, 119 mmol, 2.75 equiv) was added. The reac-
tion mixture was stirred under a nitrogen atmosphere overnight. The
reaction was cooled to 0 °C and H2O (100 mL) and Et2O (400 mL)
Synthesis 2009, No. 1, 143–147 © Thieme Stuttgart · New York

146
R. S. Erdmann, H. Wennemers
PAPER
were added. The mixture was washed with sat. NaHCO3 (3 × 150
mL) and 1 M HCl (3 × 150 mL), then the organic layer was dried
over MgSO4 and all volatiles were removed under reduced pressure.
The slightly yellow residue was subjected to column chromatogra-
phy (SiO2; EtOAc–pentane, 1:1→2:1) to give 3.
HRMS-ESI: m/z calcd for C43H47N3O7Si+Na: 768.3080; found:
768.3087 [M + Na]+.
Collagen Model Peptide 6
The following operations were automated on a Peptide Synthesizer.
Rink amide resin (150 mg, 0.36 mmol/g) was Fmoc deprotected by
treatment with 40% piperidine in DMF for 3 min followed by treat-
ment with 20% piperidine in DMF for 10 min and washed (5×) with
DMF (Protocol A). Building block 4 (4.5 equiv) was coupled on the
resin using HCTU (4.5 equiv) and i-Pr2NEt (13.5 equiv) for 90 min
and washed (5×) with DMF (Protocol B). Protocols A and B were
repeated seven times, followed by a further Fmoc deprotection
(Protocol A) and acetylation with Ac2O (30 equiv) and i-Pr2NEt (30
equiv) in CH2Cl2 for 60 min.
Yield: 30.0 g (93%); white solid; Rf = 0.30 (EtOAc–pentane, 3:2;
UV and KMnO4).
1H NMR (400 MHz, DMSO-d6): d (major conformer) = 8.37 (t,
J = 5.9 Hz, 1 H, NHGly), 7.67–7.18 (m, 20 H, Ph2Si, Cbz, Bn),
5.14–4.86 [m, 4 H, PhCH2O(CO)N, PhCH2OCO], 4.64–4.19 (m,
3 H, HaPro, HaHyp, HgHyp), 3.96–3.23 (m, 6 H, HaGly, HdPro, Hd-
Hyp), 2.35–1.68 (m, 6 H, HbPro, HbHyp, HgPro), 0.94 (s, 9 H, t-
BuSi). Isolated signals of minor conformers: d = 8.61 (t, J = 5.9 Hz,
1 H, NHGly), 8.55 (t, J = 5.8 Hz, 1 H, NHGly), 1.03, 1.00, 0.99 (s,
9 H, t-BuSi).
13C NMR (100 MHz, DMSO-d6): d = 171.7, 171.6, 170.2, 170.0,
169.5 (amides, ester), 153.7, 153.5 (carbamate), 137.0, 135.7,
135.1, 135.0, 133.0, 132.9, 132.8, 129.9, 128.3, 128.1, 128.0, 127.9,
127.8, 127.6, 127.4, 127.0, 126.8 (Ph), 71.6, 71.3 (CgHyp), 65.7,
65.6 (Cbz, Bn), 58.0, 57.9, 57.3 (CaPro, CaHyp), 54.4, 54.1 (CdH-
yp), 46.8, 46.2 (CdPro), 40.5 (CaGly), 37.7, 37.4 (CbHyp), 29.3,
28.4 (CbPro), 26.5 (TBDPS), 23.6, 22.7 (CgPro), 18.6, 18.5 (TB-
DPS).
The resin-bound CMP 6 was washed with DMF and agitated in 1 M
TBAF in THF (5 mL) overnight. After washing with THF (5×), a
solution of TFA–CH2Cl2 (2:1, 4 mL) was added. The mixture was
agitated for 60 min and the filtrate was concentrated under reduced
pressure. The residue was dissolved in the smallest possible amount
of CH2Cl2 then Et2O was added to precipitate the peptide. The pre-
cipitate was dried under reduced pressure after centrifugation and
decantation to obtain a slightly yellow material (35 mg, quant). The
crude material was purified with RP-HPLC [gradient: 91→85.9%
of solvent B (0.1% TFA, 1% MeCN in H2O) in solvent A (MeCN)
over 30 min] to obtain CMP 6 (10 mg, 29% yield with respect to the
original resin loading) in a purity of ≥99% as a white solid.
HRMS-ESI: m/z calcd for C43H49N3O7Si+Na: 770.3237; found:
770.3261 [M + Na]+.
RP-HPLC: tR = 14.8 min (gradient: 91→85.9% of solvent B in sol-
vent A over 30 min at a flow rate of 1 mL/min).
Fmoc-Pro-Hyp(TBDPS)-Gly-OH (4)
Under a nitrogen atmosphere, MeOH (15 mL) was added to 10%
(w/w) Pd/C (300 mg, 10% w/w) followed by peptide 3 (3.00 g, 4.01
mmol, 1.0 equiv). The suspension was stirred and ammonium for-
mate (2.02 g, 32.1 mmol, 8.0 equiv) was added whereupon the mix-
ture evolved a gas. After 3 h, the mixture was filtered through a
glass frit, washed with MeOH (3 × 15 mL) and the solvent was re-
moved under reduced pressure to obtain a white foam. The foam
was dissolved in a mixture of MeCN (4 mL), H2O (4 mL) and Et3N
(1.10 mL, 8.02 mmol, 2.0 equiv). Fmoc-Cl (1.22 g, 3.61 mmol 0.9
equiv) was added and a pH of 10 was secured. The suspension was
stirred at r.t. for 30 min, diluted with CH2Cl2 (30 mL) and filtered.
The filtrate was acidified with 1 M HCl (80 mL) and extracted with
CH2Cl2 (3 × 30 mL). The combined organic layers were dried over
MgSO4 and all volatiles were removed under reduced pressure. The
oily residue was subjected to column chromatography (CH2Cl2–
MeOH–AcOH, 100:5:2) to yield 4 after co-evaporation with tolu-
ene (3×) to remove traces of AcOH.
ESI-MS: m/z calcd for C84H122N22O28: 1886.9; found: 1910.0 [M +
Na]+ (100%).
Acknowledgment
This work was supported by the Swiss National Science Foundation
and Bachem. H.W. is grateful to Bachem for an endowed professor-
ship.
References
(1) For reviews, see: (a) Brodsky, B.; Thiagarajan, G.; Madhan,
B.; Kar, K. Biopolymers 2008, 89, 345. (b) Raines, R. T.
Protein Sci. 2006, 15, 1219. (c) Engel, J.; Baechinger, H. P.
Top. Curr. Chem. 2005, 247, 7. (d) Fields, G. B.; Prockop,
D. J. Biopolymers 1996, 40, 345.
Yield: 2.31 g (86%); white foam; Rf = 0.29 (CH2Cl2–MeOH–
AcOH, 100:5:2; UV). HPLC: tR = 26.1 min [gradient: 50→10% of
solvent B (0.1% TFA, 1% MeCN in H2O) in solvent A (MeCN)
over 35 min at a flow rate of 1 mL/min].
1H NMR (500 MHz, CD3CN): d = 7.95–7.75, 7.70–7.53, 7.51–7.28
(m, 18 H, Fmoc, TBDPS), 7.24, 7.04 (t, J = 5.6 Hz, 1 H, NHGly),
4.63–4.35 (m, 2 H, HaHyp, HaPro), 4.58–4.48 (m, 1 H, HgHyp),
4.50–4.33 (m, 2 H, Fmoc), 4.32–4.18 (m, 1 H, Fmoc), 3.90–3.75
(m, 2 H, HaGly), 3.54–3.11 (m, 4 H, HdHyp, HdPro), 2.22–1.91 (m,
2 H, HbHyp), 2.15–2.05, 1.88–1.64 (m, 2 H, HbPro), 1.88–1.64 (m,
2 H, HgPro), 1.04, 1.02, 0.92 (3 × s, 9 H, t-BuSi).
13C NMR (125 MHz, CD3CN): d = 173.0, 172.7, 172.6, 171.2
(amides, carboxylic acid), 155.5, 155.2 (carbamate), 145.3, 145.2,
145.1, 142.2, 142.1, 142.0, 138.9, 136.5, 136.4, 134.5, 134.2, 131.0,
128.9, 128.8, 128.7, 128.6, 128.2, 128.1, 126.1, 126.0, 120.9 (Fmoc,
TBDPS), 73.0, 72.6 (CgHyp), 67.6, 67.5 (Fmoc), 60.2, 59.6, 58.9
(CaPro, CaHyp), 55.6, 54.8 (CdHyp), 48.2, 48.1 (Fmoc), 48.0, 47.3
(CdPro), 41.7, 41.6 (CaGly), 38.0, 37.6 (CbHyp), 30.7, 29.7 (CbPro),
27.2, 27.1 (TBDPS), 24.9, 23.7 (CgPro), 19.6, 19.5 (TBDPS). Sig-
nals were assigned by 2-D NMR spectroscopy (COSY, HMBC and
HMQC).
(2) Peterkofsky, B. Am. J. Clin. Nutr. 1991, 54, 1135.
(3) Persikov, A. V.; Ramshaw, J. A. M.; Brodsky, B.
Biopolymers 2000, 55, 436.
(4) For examples, see: (a) Lee, S.-G.; Lee, J. Y.; Chmielewski,
J. Angew. Chem. Int. Ed. 2008, 47, 8429. (b) Dai, N.;
Wang, X. J.; Etzkorn, F. A. J. Am. Chem. Soc. 2008, 130,
5396. (c) Gauba, V.; Hartgerink, J. D. J. Am. Chem. Soc.
2008, 130, 7509. (d) Gauba, V.; Hartgerink, J. D. J. Am.
Chem. Soc. 2007, 129, 2683. (e) Gauba, V.; Hartgerink, J.
D. J. Am. Chem. Soc. 2007, 129, 15034. (f) Bachmann, A.;
Kiefhaber, T.; Boudko, S.; Engel, J.; Bachinger, H. P. Proc.
Natl. Acad. Sci. U.S.A. 2005, 102, 13897. (g) Beck, K.;
Chan, V. C.; Shenoy, N.; Kirkpatrick, A.; Ramshaw, J. A.
M.; Brodsky, B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
4273.
(5) For examples of CMPs where some or all Hyp residues are
replaced by other amino acids, see: (a) Kotch, F. W.; Guzei,
I. A.; Raines, R. T. J. Am. Chem. Soc. 2008, 130, 2952.
(b) Cadamuro, S. A.; Reichold, R.; Kusebauch, U.; Musiol,
H.-J.; Renner, C.; Tavan, P.; Moroder, L. Angew. Chem. Int.
Ed. 2008, 47, 2143. (c) Raines, R. T. Protein Sci. 2006, 15,
Synthesis 2009, No. 1, 143–147 © Thieme Stuttgart · New York

PAPER
1219. (d) Kusebauch, U.; Cadamuro, S. A.; Musiol, H.-J.;
Efficient Synthesis of Collagen Model Peptides
147
Maryanoff, B. E. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
8513. (b) Przybyla, D. E.; Chmielewski, J. J. Am. Chem.
Soc. 2008, 130, 12610. (c) Rele, S.; Song, Y.; Apkarian, R.
P.; Qu, Z.; Conticello, V. P.; Chaikof, E. L. J. Am. Chem.
Soc. 2007, 129, 14780. (d) Cejas, M. A.; Kinney, W. A.;
Chen, C.; Leo, G. C.; Tounge, B. A.; Vinter, J. G.; Joshi, P.
P.; Maryanoff, B. E. J. Am. Chem. Soc. 2007, 129, 2202.
(e) Kar, K.; Amin, P.; Bryan, M. A.; Persikov, A. V.; Mohs,
A.; Wang, Y.-H.; Brodsky, B. J. Biol. Chem. 2006, 281,
33283. (f) Kotch, F. W.; Raines, R. T. Proc. Natl. Acad. Sci.
U.S.A. 2006, 103, 3028.
Lenz, M. O.; Wachtveitl, J.; Moroder, L.; Renner, C. Angew.
Chem. Int. Ed. 2006, 45, 7015. (e) Hodges, J. A.; Raines, R.
T. J. Am. Chem. Soc. 2005, 127, 15923. (f) Jenkins, C. L.;
McCloskey, A. I.; Guzei, I. A.; Eberhardt, E. S.; Raines, R.
T. Biopolymers 2005, 80, 1. (g) Hodges, J. A.; Raines, R. T.
J. Am. Chem. Soc. 2003, 125, 9262. (h) Jenkins, C. L.;
Bretscher, L. E.; Guzei, I. A.; Raines, R. T. J. Am. Chem.
Soc. 2003, 125, 6422. (i) Babu, I. R.; Ganesh, K. N. J. Am.
Chem. Soc. 2001, 123, 2079. (j) Bretscher, L. E.; Jenkins, C.
L.; Taylor, K. M.; DeRider, M. L.; Raines, R. T. J. Am.
Chem. Soc. 2001, 123, 777. (k) Holmgren, S. K.; Bretscher,
L. E.; Taylor, K. M.; Raines, R. T. Chem. Biol. 1999, 6, 63.
(l) Holmgren, S. K.; Taylor, K. M.; Bretscher, L. E.; Raines,
R. T. Nature 1998, 392, 666.
(7) (a) Ottl, J.; Musiol, H. J.; Moroder, L. J. Pept. Sci. 1999, 5,
103. (b) Inouye, K.; Sakakibara, S.; Prockop, D. J. Biochim.
Biophys. Acta 1976, 420, 133. (c) Sakakibara, S.; Inouye,
K.; Shudo, K.; Kishida, Y.; Kobayashi, Y.; Prockop, D. J.
Biochim. Biophys. Acta 1973, 303, 198.
(8) Bodansky, M. Principles of Peptide Synthesis, 2nd ed.;
Springer Laboratory: Heidelberg, 1993, 135.
(6) For examples of supramolecular assemblies formed by
CMPs, see: (a) Cejas, M. A.; Kinney, W. A.; Chen, C.;
Vinter, J. G.; Almond, H. R. Jr.; Balss, K. M.; Maryanoff, C.
A.; Schmidt, U.; Breslav, M.; Mahan, A.; Lacy, E.;
(9) Sajiki, H.; Ikawa, T.; Hattori, K.; Hirota, K. Chem. Commun.
2003, 654.
Synthesis 2009, No. 1, 143–147 © Thieme Stuttgart · New York