The design, synthesis, and initial evaluation of benzophenone-containing peptides as potential photoaffinity labels of oligosaccharyltransferase

Article abstract of DOI:10.1016/S0968-0896(98)00135-7

The benzophenone photophore was incorporated into protected tripeptides and tetrapeptides as photoactivatable probes to study the multimeric enzyme oligosaccharyltransferase (OST). These peptides contain the -Asn-X-Thr- sequon which is required for OST-ca

Full text of DOI:10.1016/S0968-0896(98)00135-7

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 1821±1834
The Design, Synthesis, and Initial Evaluation of Benzophenone-
containing Peptides as Potential Photoanity Labels of
Oligosaccharyltransferase
Tong Xu, Hemant Khanna and James K. Coward*
Department of Chemistry, and Interdepartmental Program of Medicinal Chemistry, College of Pharmacy, The University of Michigan, Ann
Arbor, Michigan 48109, USA
Received 25 March 1998; accepted 14 May 1998
AbstractÐThe benzophenone photophore was incorporated into protected tripeptides and tetrapeptides as photo-
activatable probes to study the multimeric enzyme oligosaccharyltransferase (OST). These peptides contain the -Asn-
X-Thr- sequon which is required for OST-catalyzed N-glycosylation. Two tripeptides, Bz-Asn-Bpa-Thr-NH2 (3b) and
Bz-Asn-Lys[Ne-(4-Bz)Bz]-Thr-NH2 (4b), were found to be good OST substrates. They were competitive inhibitors
versus standard peptide substrate [14C]Bz-Asn-Leu-Thr-NH2 and their Ki values were determined to be 41‹6 mM and
21‹6 mM, respectively, using synthetic (GlcNAc)2-PP-dolichol. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
imide,7 enol lactone,8 and ketene8 intermediates were
investigated and found to be unlikely. Another pro-
posed catalytic mechanism, similar to that of many glut-
amine-dependent amidotransferases, was recently
Oligosaccharyltransferase1,2 (OST, EC 2.4.1.119) is the
key enzyme in the N-linked glycoprotein synthesis. It
transfers the oligosaccharide portion of Glc3Man9Glc-
NAc2-PP-dolichol (Dol-PP-OS) to the side chain amide
moiety of an asparagine residue that is part of the -Asn-
X-Ser/Thr- sequon3,4 in a growing polypeptide chain
(Fig. 1). During the cotranslational process, the seem-
ingly non-nucleophilic side chain carboxamido group
e€ects a SN2-like nucleophilic attack at the anomeric
carbon of the ®rst GlcNAc and displaces the dolichol
pyrophosphate. Mechanistic studies of this intriguing
reaction have been actively pursued during the past ®f-
teen years. Several mechanisms have been proposed to
address activation of the carboxamido moiety of aspar-
agine during OST catalysis such as the active site base
deprotonation mechanism proposed by Bause et al.5
and the peptide `Asx-turn' conformation activation
mechanism proposed by Imperiali and co-workers.1,6
Three nucleophilic activation mechanisms involving
studied using 13C/15N-labeled peptide substrates.9
A
lack of `NH3' exchange suggested no similarity between
the OST catalytic mechanism and that of the glutamine-
dependent amidotransferases.
Based on the fact that methyl methanethiolsulfonate
(MMTS)10,11 and p-chloromercury benzoate (pCMB)
(Liu, Y. L.; Schretzman, L.; Coward, J. K., unpublished
results) inhibit OST-catalyzed glycosylation, it has been
postulated that a crucial cysteine residue may be
involved in catalysis. However, inhibition by nonspeci®c
group reagents does not address the actual roles of this
seemingly important cysteine residue in OST catalysis.
Although Bause12,13 has demonstrated that a heptapep-
tide containing -Asn-Gly-(epoxyethyl)Gly- sequon
covalently labels OST, our recent work has shown that
peptides with asparagine replaced by structurally similar
amino acids could not act as e€ective anity labels or
competitive inhibitors of OST.11 These results seemed to
indicate that the important cysteine may not be located
in the enzyme active site and it is less likely to play a
major role in catalysis. This assumption is further sup-
ported by the fact that (GlcNAc)2-PP-dolichol (Dol-PP-
Key words: Benzophenone; photophore; photoanity label;
oligosaccharyltransferase.
*Corresponding author. Tel: (734) 936-2843; Fax: (734) 647-
4865.
0968-0896/98/$Ðsee front matter # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(98)00135-7

1822
T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
Figure 1. OST-catalyzed N-linked glycoprotein synthesis.
DS) protected OST from being inhibited by MMTS,10,11
an indication that cysteine could be involved in glyco-
lipid substrate binding. Therefore, mechanistic probes
which target less speci®c groups on the protein need to
be developed.
approach would enable us to identify the critical sub-
unit(s) via the synthesis of alternate substrates that
would carry photophores capable of labeling the active
site when photoirradiated.
Application of the benzophenone photophore as a bio-
chemical probe has increased dramatically during the
past ten years.19 Compared with an aryl azide, a benzo-
phenone is chemically more stable, can be activated at
longer wavelength (350±360 nm), and preferentially
inserts into a C±H bond, even in aqueous solution or in
the presence of other nucleophiles. If the benzophenone
photophore is incorporated into peptides which contain
the -Asn-X-Ser/Thr- sequon, these peptides, when pho-
toactivated, should act as photoanity labels of OST.
Four peptides have been designed for this purpose. They
are (4-Bz)Bz-Asn-Leu-Thr-NH2 (1b), Bz-Bpa-Asn-Leu-
Thr-NH2 (2b), Bz-Asn-Bpa-Thr-NH2 (3b), and Bz-Asn-
Lys[Ne-(4-Bz)Bz]-Thr-NH2 (4b). The requirement of
-Asn-X-Ser/Thr- sequon is satis®ed in all four peptides.
Nonphotolabile control peptides (1±4a), in which the
benzophenone photophore of the four potential photo-
anity labels (1±4b) is replaced by a phenyl group, have
also been synthesized.
In the 1980s, Lennarz and co-workers developed 3H-
and 125I-labeled, aryl azide-containing tripeptides as
active site-directed photoanity labels of OST.14±16 The
photophore was tethered to the side chain e-amino
group of lysine in the -Asn-Lys-Thr- sequon. However,
photoinhibition studies using microsomal OST enzyme
showed that the protein covalently labeled was not
membrane-bound but an enzyme located in the lumen
of the endoplasmic reticulum.17 Further research indi-
cated that the labeled protein was protein disul®de iso-
merase18 which was not required for N-linked
glycoprotein synthesis. In the intervening years, OST
has been successfully puri®ed from ®ve di€erent sources,2
suggesting that the photoanity label approach to study
OST catalysis is now amenable to reinvestigation. OST
is a complicated multisubunit enzyme with limited
information available as to which subunits comprise its
active site. Application of the photoanity label

T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
1823
Results and Discussion
amino group23 (Scheme 3). Decomposition of the cop-
per chelate complex 11 by thioacetamide followed by
the protection of the a-amino group with Boc2O a€or-
ded 9a. The synthesis of 9b started with Na-Boc-Ne-Cbz-
Lys. 2-Trimethylsilylethyl (TMSE) ester formation24
followed by hydrogenolysis gave Na-Boc-Lys-
OTMSE.25 Coupling of Na-Boc-Lys-OTMSE with 4-
benzoylbenzoic acid followed by the deprotection of the
TMSE ester a€orded 9b.
Synthesis
Compound 1b was synthesized by the coupling of H-
.
Asn-Leu-Thr-NH2 TFA with 4-benzoylbenzoic acid
(5b) using EDC (Scheme 1). N-Boc-Phe (6a) or N-Boc-
.
Bpa (6b) was coupled with H-Asn-Leu-Thr-NH2 TFA
to a€ord tetrapeptides 7a and 7b using standard solu-
tion phase carbodiimide coupling method. Deprotection
at the N-terminal by TFA followed by the benzoylation
gave the tetrapeptide 2. Tripeptides 3a and 3b were
synthesized in the similar fashion (Scheme 2). The
synthesis of lysine-containing tripeptide 4 is more
involved. Initially, Na-Boc-Ne-Cbz-Lys was treated with
diazomethane to give the methyl ester. Na-Boc-Ne-Cbz-
Lys-OMe was then treated under hydrogenolysis and
the resulting free e-amino group was coupled with 4-
benzoylbenzoic acid (5b). The resulting lysine analogue
was treated with TFA to remove the Na-Boc group and
the resulting free a-amine was coupled with N-Boc-Asn.
Unfortunately, the resulting Boc-Asn-Lys[Ne-(4-Bz)Bz]-
OMe dipeptide was resistant to hydrolysis when treated
with K2CO3 in aq DMF. The successful synthesis of 4
was accomplished using Na-Boc-Ne-Bz-Lys (9a)20 and
Na-Boc-Ne-[(4-Bz)Bz]-Lys (9b)21 (Scheme 2). The synth-
esis procedure was similar to that of 3. The synthesis of
9a involved formation of the lysine-copper chelate22
followed by the selective benzoylation of the lysine e-
Biochemical studies
The four potential photoanity labels (1±4b) and the
corresponding control peptides (1±4a) were ®rst eval-
uated as peptide substrates for OST using microsomal
enzyme and either [3H]Dol-PP-OS or [3H]Dol-PP-
DS.7,26 The results shown in Table 1 are independent of
the saccharide donor; i.e. [3H]Dol-PP-OS or [3H]Dol-
PP-DS lead to the same conclusions. The tetrapeptide
2b is not a substrate while 1b is a modest acceptor sub-
strate. However, the other two tripeptides 3b and 4b, in
which the middle amino acid is Bpa or Lys analogue,
are good peptide substrates of OST. These two tripep-
tides were further evaluated as inhibitors (alternate
substrates) versus the standard peptide substrate Bz-
Asn-Leu-Thr-NH2 (1a) using synthetic Dol-PP-DS.26,27
Dixon plots28 (1/V versus [I] at various substrate con-
centration) led to Ki values for 3b and 4b of 41‹6 mM

1824
T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
and 21‹6 mM, respectively (Fig. 2). These values are
considerably lower than the Km of Bz-Asn-Leu-Thr-
NH2 (282 mM) (Gibbs, B. S.; Coward, J. K., unpub-
lished results), perhaps due to the hydrophobic environ-
ment at the glycosylation site at the rough endoplastic
reticulum membrane.
dard substrate Bz-Asn-Leu-Thr-NH2 (1a) and their Ki
values were found to be 41‹6 mM and 21‹6 mM,
respectively. The photoanity approach is a useful tool
that can be applied toward the understanding of the
complicated OST enzyme. This approach to identifying
the OST active site appears more promising following
puri®cation of this multimeric enzyme in recent years.
Initial photochemical studies showed that 3b and 4b are
photolabile and the absorbance at 262 nm decreases
during photolysis. However, control peptides 3a and 4a,
lacking the benzophenone photophore, did not show
this characteristic. Further photochemical evaluations
of 3b and 4b in the OST enzyme system and the synth-
esis of radiolabeled 3b and 4b are currently in progress.
Experimental
General
NMR Spectra were obtained with Bruker instruments
1
operating at 360 MHz or 300 MHz. H and 13C NMR
chemical shifts were reported in ppm down®eld from
TMS. Melting points were uncorrected. Infrared spectra
were recorded as a thin ®lm on sodium chloride plates
or as a KBr pellet, and absorptions were reported in
wavenumbers (cm 1). MS, HRMS, and elemental ana-
lysis were performed in the Microanalytical Facility,
Department of Chemistry, University of Michigan. TLC
Conclusion
Two protected tripeptides, Bz-Asn-Bpa-Thr-NH2 (3b)
and Bz-Asn-Lys[Ne-(4-Bz)Bz]-Thr-NH2 (4b), were
found to be good OST substrates. They were also good
competitive inhibitors (alternate substrates) versus stan-
Scheme 1.

T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
1825
spots were visualized by UV, ninhydrin, or bromocresol
green spray reagents. THF was freshly distilled from
sodium benzophenone ketyl, while dichloromethane and
N-methylmorpholine (NMM) were distilled from CaH2.
N-Boc-Bpa was bought from Bachem, N-Boc-Phe and
4-benzoylbenzoic acid were from Aldrich, and Na-Boc-
Ne-Cbz-Lys was purchased from Sigma. All other
reagents were purchased from major commercial sour-
ces and were used without further puri®cation. H-Asn-
viously described.7 (GlcNAc)2-PP-dolichol was synthe-
sized as described by Lee and Coward,26 and its
concentration was determined by high pH anion-
exchange chromatography (HPAEC)29 following
hydrolysis to glucosamine. Ne-Bz-l-Lysine (12)23 and
Na-Boc-Ne-Cbz-l-lysine 2-trimethylsilylethyl ester (13)24
were prepared as described in the literature. P40 yeast
microsomes and [3H](GlcNAc)2-PP-dolichol were pre-
pared as described by Clark et al.7 and Lee and Coward,26
respectively. Standard OST assay used to measure the
.
Leu-Thr-NH2 TFA and 1a were synthesized as pre-
Scheme 2.

1826
T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
peptide acceptor ability was described previously.7,26
Enzyme assays used for Ki determination were analyzed
by reverse phase HPLC on a Waters liquid chromato-
graphy systems (6100A & 510 pumps), Rainin Micro-
sorb±MV 5 mM C18, 300 A, 4.6Â250 mm column, and
monitored using a Waters 996 diode array spectrometer.
Radioactivity was determined using a Packard 1600 TR
liquid scintillation analyzer.
0.22 mmol), and H-Asn-Leu-Thr-NH2 TFA (0.20 mmol)
.
were dissolved in 2 mL dry DMF at 0 ꢀC while stirring
and NMM (46 mL, 0.42 mmol) was added. The reaction
mixture was stirred at 0 ꢀC for 1 h and at rt for 16 h. The
reaction mixture was concentrated in vacuo and the
resulting brown residue was triturated with 5% citric
acid solution. The white precipitate was collected,
washed with cold water, and then crystallized from
water/acetone/methanol (v/v/v, 1/1/1). 90 mg (87%) of a
white solid was obtained: Rf 0.65 (MeOH/EtOAc, 1/1);
N-(4-Bz)Bz-Asn-Leu-Thr-NH2 (1b). 4-Benzoylbenzoic
.
acid (5b, 45 mg, 0.20 mmol), EDC HCl (42 mg,
mp 233±235 ꢀC (dec); H NMR (DMSO-d6) d 0.86 (dd,
1
Scheme 3.

T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
1827
6H), 1.03 (d, 3H), 1.52 (m, 2H), 1.66 (m, 1H), 2.68 (m,
2H), 4.04 (m, 1H), 4.08 (m, 1H), 4.32 (m, 1H), 4.81 (m,
1H), 4.92 (d, 1H), 6.95±7.15 (m, 4H), 7.50±8.05 (m, 9H),
7.68 (d, 1H), 8.31 (d, 1H), 8.92 (d, 1H); 13C NMR
(DMSO-d6) d 19.9, 21.4, 23.0, 24.1, 36.8, 40.3, 51.0,
51.7, 58.3, 66.3, 127.2, 128.4, 129.1, 129.4, 132.8, 136.5,
137.2, 139.2, 165.4, 170.9, 171.6, 171.7, 172.0, 195.2; MS
(FAB+) m/e (rel. intensity) 554 (MH+, 44), 209 (76), 86
(100); HRMS calcd for C28H36N5O7 (MH+), 554.2615;
.
found, 554.2587. Anal. calcd for C28H35N5O7 1.2H2O:
C, 58.46; H, 6.55; N, 12.17. Found: C, 58.36; H, 6.35; N,
12.02.
Table 1. Evaluation of photophore-containing peptides as OST substratesa
Relative glycosylation activityb
Compd
R
X
[3H]LOS
[3H]LDS
1a
(CH3)2CHCH2-
1.00
1.00
1b
2a
(CH3)2CHCH2-
(CH3)2CHCH2-
0.38
0.24
0.26
0.23
2b
(CH3)2CHCH2-
0
0.06
3a
3b
4a
1.04
1.00
1.23
1.11
1.09
1.30
4b
0.86
1.04
aMicrosomal OST enzyme was used.
bRelative to glycosylation of Bz-Asn-Leu-Thr-NH2 (1.00).

1828
T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
2.50±3.15 (m, 4H), 4.0±4.5 (m, 5H) 7.17 (m, 5H); 13C
NMR (DMSO-d6, CD3OD) d 20.8, 22.0, 24.0, 25.9,
29.0, 37.6, 39.0, 41.4, 51.8, 54.0, 57.6, 60.7, 68.3, 80.7,
127.9, 129.6, 130.6, 139.0, 157.7, 173.4, 174.3, 174.77,
174.81, 175.2; MS (FAB+) m/e (rel. intensity) 593
(MH+, 28), 120 (72), 86 (100); HRMS calcd for
C28H45N6O8 (MH+), 593.3299; found, 593.3307.
N-Bz-Phe-Asn-Leu-Thr-NH2 (2a). N-Boc-Phe-Asn-Leu-
Thr-NH2 (7a, 70 mg, 0.12 mmol) was dissolved in 2 mL
TFA/CH2Cl2 (v/v, 1/1) at rt and the mixture was
allowed to set at rt for 30 min. The reaction mixture was
concentrated in vacuo and the resulting clear oil was
triturated with Et2O. The precipitate was collected and
rinsed with Et2O and air dried to give 71 mg white solid:
99% yield; Rf 0.31 (EtOAc/MeOH, 1/1). This product,
.
H-Phe-Asn-Leu-Thr-NH2 TFA (66 mg, 0.11 mmol), was
dissolved in 1.5 mL H2O/dioxane/DMF (v/v/v, 1/1/1)
mixed solvent. Et3N (15 mL, 0.11 mmol) was added at
0 ꢀC followed by Bz2O (37 mg, 0.16 mmol) while stirring.
The mixture was stirred for 5 min then Et3N (8 mL,
0.06 mmol) was further added. The reaction mixture was
stirred at rt for 12 h and then concentrated in vacuo.
The resulting residue was dissolved in H2O/THF (v/v, 1/
1) and Dowex 50W-X8 (H+) resin was added and the
mixture was gently stirred. The ®ltrate was concentrated
in vacuo and the resulting crude product was crystal-
lized from H2O/THF (v/v, 1/1), rinsed with Et2O, and
dried to give 57 mg (88%) of a white solid: Rf 0.69
(MeOH/EtOAc, 1/1); mp 247±249 ꢀC; 1H NMR
(DMSO-d6) d 0.80 (dd, 6H), 1.01 (d, 3H), 1.51 (m, 2H),
1.60 (m, 1H), 2.55±3.15 (m, 4H), 4.02 (m, 1H), 4.04 (m,
1H), 4.28 (m, 1H), 4.57 (m, 1H), 4.69 (m, 1H), 6.95±7.80
(m, 14H), 7.62, 8.06, 8.51, 8.61 (d, 4H); 13C NMR
(DMSO-d6) d 20.1, 21.3, 23.2, 24.1, 36.7, 37.0, 40.2,
49.9, 51.5, 55.0, 58.5, 66.4, 126.3, 127.4, 128.1, 128.2,
129.2, 131.3, 134.0, 138.4, 166.5, 171.0, 171.5, 172.0,
172.3, 174.9; MS (FAB+) m/e (rel intensity) 597 (MH+,
34), 336 (40), 252 (42), 224 (41), 105 (100), 85 (68);
HRMS calcd for C30H41N6O7 (MH+), 597.3037; found,
Figure 2. Dixon plot of OST inhibition by (A) compound 3b,
(B) compound 4b. [[14C]Bz-Asn-Leu-Thr-NH2]=90(^), 180
(*), and 360 (&) mM.
N-Boc-Phe-Asn-Leu-Thr-NH2 (7a). N-Boc-Phe (6a,
.
89 mg, 0.34 mmol), EDC HCl (78 mg, 0.40 mmol), and
.
597.3037. Anal. calcd for C30H40N6O7 0.5H2O: C,
HOBt (68 mg, 0.51 mmol) were dissolved in 1 mL anhy-
drous DMF at 10 ꢀC while stirring. NMM (44 mL,
0.41 mmol) was added followed by a precooled solution
59.50; H, 6.78; N, 13.88. Found: C, 59.58; H, 6.74; N,
13.40.
.
of H-Asn-Leu-Thr-NH2 TFA (0.34 mmol) and NMM
N-Boc-Bpa-Asn-Leu-Thr-NH2 (7b). N-Boc-Bpa (6b,
.
124 mg, 0.34 mmol), EDC HCl (78 mg, 0.41 mmol), and
HOBt (68 mg, 0.51 mmol) were dissolved in 1 mL dry
DMF. NMM (44 mL, 0.41 mmol) was added at 10 ꢀC
and the mixture was stirred for 10 min under nitrogen.
(37 mL, 0.34 mmol) in 1.5 mL anhydrous DMF. The
reaction mixture was stirred at 10 ꢀC for 1 h and at rt
for 18 h. The reaction mixture was concentrated in
vacuo and the yellow residue obtained was triturated
with 5% citric acid solution. The white precipitate was
collected and rinsed with water. The crude product was
further triturated with saturated NaHCO3 solution,
rinsed with water and dried to give 165 mg (83%) of a
white solid: Rf 0.59 (EtOAc/MeOH, 3/1); mp 220±
.
A precooled solution of H-Asn-Leu-Thr-NH2 TFA,
obtained by treatment of N-Boc-Asn-Leu-Thr-NH2
(150 mg, 0.34 mmol) with TFA,7 and NMM (37 mL,
0.34 mmol) in 1.5 mL dry DMF was added while stir-
ring. The reaction mixture was stirred at 10 ꢀC for 1 h
and at rt for 18 h and then concentrated in vacuo. The
resulting yellow residue was triturated with 5% citric
222 ꢀC; H NMR (DMSO-d6, CD3OD) d 0.80 (dd, 6H),
1
1.05 (d, 3H), 1.25 (s, 9H), 1.55 (m, 2H), 1.60 (m, 1H),

T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
1829
acid solution and the slightly yellowish precipitate
obtained was further triturated with saturated NaHCO3
solution. The precipitate was collected by ®ltration,
rinsed with H2O, and dried to give 178 mg (76%) of a
white solid: Rf 0.54 (MeOH/EtOAc, 1/3); mp 209±
211 ꢀC; IR (KBr): 3416, 3311, 3072, 2973, 2931, 1666,
at 10 ꢀC for 15 min and a precooled mixture of
.
HCl Thr-NH2 (231 mg, 1.5 mmol) and NMM (165 mL,
1.5 mmol) in 4 mL dry DMF was added. The reaction
mixture was stirred at 10 ꢀC for 1 h and at rt for 10 h.
Solvent was removed in vacuo and 6 mL 5% citric acid
solution was added to triturate the slightly brown resi-
due. The white precipitate was collected and rinsed with
water and Et2O. The crude product was further puri®ed
by Et2O trituration to give 466 mg (85%) of N-Boc-Phe-
Thr-NH2 as a white solid: Rf 0.61 (MeOH/EtOAc, 1/1);
mp 154±155 ꢀC; IR (KBr): 3430, 3367, 3339, 3030, 2981,
1
1525, 1277,702 cm 1; H NMR (DMSO-d6) d 0.87 (m,
6H), 1.10 (d, 3H), 1.34 (s, 9H), 1.62 (m, 2H), 1.73 (m,
1H), 2.55±3.20 (m, 4H), 4.10±4.75 (m, 5H), 6.85±7.05
(m, 4H), 7.35±8.40 (m, 13H); 13C NMR (DMSO-d6) d
20.2, 21.4, 23.2, 24.2, 28.2, 37.0, 37.6, 40.3, 49.9, 51.7,
55.4, 56.7, 58.6, 66.4, 78.3, 128.6, 129.5, 132.6, 135.1,
137.3, 143.6, 155.3, 171.1, 171.4, 172.0, 172.3, 172.9,
195.6; MS (FAB+) m/e (rel. intensity) 697 (MH+, 28),
224 (30), 87 (42), 86 (100); HRMS calcd for
C35H49N6O9 (MH+), 697.3561; found, 697.3589.
1
2938, 1687, 1659, 1525, 1166 cm 1; H NMR (DMSO-
d6) d 1.02 (d, 2H), 1.30 (s, 9H), 2.60±3.10 (m, 2H), 4.07
(m, 1H), 4.10 (m, 1H), 4.17 (m, 1H), 4.95 (d, 1H), 7.08±
7.20 (m, 3H), 7.26 (m, 5H), 7.57 (d, 1H); 13C NMR
(DMSO-d6) d 19.9, 28.2, 36.9, 56.1, 57.8, 66.3, 78.4,
126.1, 128.0, 129.1, 138.1, 155.3, 171.6, 171.9.
N-Bz-Bpa-Asn-Leu-Thr-NH2 (2b). N-Boc-Bpa-Asn-Leu-
Thr-NH2 (7b, 80 mg, 0.11 mmol) was dissolved in 2 mL
TFA/CH2Cl2 (v/v, 1/1) and the mixture was allowed to
set at rt for 30 min. The clear solution was concentrated
in vacuo and the resulting colorless oil was triturated
with Et2O. The white precipitate was collected and
rinsed with Et2O and air dried to give 81 mg white solid,
99% yield. Rf 0.36 (MeOH/EtOAc, 1/1). H-Bpa-Asn-
N-Boc-Phe-Thr-NH2 (292 mg, 0.80 mmol) was dissolved
in 5 mL TFA/CH2Cl2 (v/v, 1/1) at rt and the mixture
was stirred for 30 min. The solvent was removed in
vacuo and the resulting clear oil was triturated with
Et2O. The white precipitate was collected, dried, and
directly used for the following reaction. Rf 0.51 (MeOH/
EtOAc, 1/1). N-Boc-Asn (186 mg, 0.80 mmol), DCC
(198 mg, 0.96 mmol), and HOBt (162 mg, 1.2 mmol)
.
Leu-Thr-NH2 TFA (74 mg, 0.10 mmol) was dissolved in
1.5 mL H2O/dioxane/DMF (v/v/v, 1/1/1) mixed solvent.
Et3N (14 mL, 0.10 mmol) was added at 0 ꢀC followed by
Bz2O (35 mg, 0.16 mmol) while stirring. The reaction
mixture was stirred for 5 min and Et3N (7 mL,
0.05 mmol) was further added. The reaction mixture was
stirred at rt for 12 h and then concentrated in vacuo.
The resulting residue was dissolved in H2O/THF (v/v, 1/
1) and Dowex 50W X8 (H+) resin was added and the
mixture was gently stirred. The mixture was ®ltered and
the ®ltrate was concentrated in vacuo. The resulting
white residue was crystallized from H2O/THF (v/v, 1/1).
The crude product was rinsed with Et2O and dried:
63 mg; 86% yield; Rf 0.72 (MeOH/EtOAc, 1/1); mp
were dissolved in 5 mL dry DMF at 0 ꢀC while stirring.
.
A
precooled solution of H-Phe-Thr-NH2 TFA
(0.80 mmol) and NMM (88 mL, 0.80 mmol) in 4 mL dry
DMF was added. The reaction mixture was stirred at
0 ꢀC for 1 h and at rt for 16 h. The precipitate was ®l-
tered and the ®ltrate was concentrated in vacuo. The
resulting yellow residue was triturated with EtOAc/Et2O
(v/v, 1/1). The precipitate collected was further tritu-
rated with EtOAc. The white precipitate was collected
and air dried to give 310 mg (81%) of the desired pro-
duct. Rf 0.70 (MeOH/EtOAc, 1/3); mp 207±209 ꢀC; H
1
NMR (DMSO-d6) d 1.01 (d, 2H), 1.36 (s, 9H), 2.38 (m,
2H), 2.80±3.10 (m, 2H), 4.03 (m, 1H), 4.08 (m, 1H), 4.23
(m, 1H), 4.56 (m, 1H), 4.83 (d, 1H), 6.93 (s, 2H), 7.05 (d,
2H), 7.22 (m, 5H), 7.31 (d, 1H), 7.84 (d, 1H), 7.94 (d,
1H); 13C NMR (DMSO-d6) d 19.9, 28.1, 33.3, 37.2, 51.3,
53.9, 58.2, 66.3, 78.2, 126.0, 127.8, 129.1, 137.4, 154.8,
170.6, 171.2, 171.5, 171.8.
244±246 ꢀC; H NMR (DMSO-d6) d 0.82 (m, 6H), 1.03
1
(d, 3H), 1.52 (m, 2H), 1.62 (m, 1H), 2.55±3.20 (m, 4H),
4.00±4.75 (m, 5H), 6.95±7.80 (m, 18H), 7.92, 8.11, 8.56,
8.67 (d, 4H); 13C NMR (DMSO-d6) d 20.1, 21.3, 23.2,
24.1, 36.8, 37.1, 40.2, 49.9, 51.6, 54.5, 58.5, 66.4, 127.4,
128.2, 128.5, 129.4, 129.5, 129.6, 131.4, 132.5, 132.9,
133.9, 135.1, 137.2, 139.2, 143.7, 166.6, 171.0, 171.2,
171.9, 171.9, 172.2, 195.5; MS (FAB+) m/e (rel inten-
sity) 701 (MH+, 27), 155 (33), 119 (52), 105 (100);
HRMS calcd for C37H45N6O8 (MH+), 701.3299; found,
N-Bz-Asn-Phe-Thr-NH2 (3a). N-Boc-Asn-Phe-Thr-NH2
(8a, 184 mg, 0.384 mmol) was dissolved in 5 mL TFA/
CH2Cl2 (v/v, 1/1) and the mixture was stirred for 30 min
at rt. Solvents were removed in vacuo and the resulting
clear oil was triturated with Et2O. The white precipitate
was collected, rinsed with Et2O, dried, and used directly
for the following reaction; Rf 0.32 (MeOH/EtOAc, 1/3).
.
701.3301. Anal. calcd for C37H44N6O8 H2O: C, 61.83;
H, 6.45; N, 11.69. Found: C, 61.81; H, 6.38; N, 11.39.
.
The product, H-Asn-Phe-Thr-NH2 TFA, obtained was
N-Boc-Asn-Phe-Thr-NH2 (8a). To a stirred solution of
N-Boc-Phe (6a, 398 mg, 1.5 mmol) in 6 mL dry THF at
10 ꢀC was added NMM (165 mL, 1.5 mmol) followed
by iBCF (215 mL, 1.65 mmol). The mixture was stirred
dissolved in 4 mL water and Et3N (60 mL, 0.38 mmol)
was added at 0 ꢀC while stirring. 4 mL dioxane and
Bz2O (96 mg, 0.422 mmol) were added and the mixture

1830
T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
was stirred at 0 ꢀC for 5 min. 30 mL Et3N was further
added. The reaction mixture was stirred at 0 ꢀC for 1 h
and at rt for 14 h and then concentrated in vacuo. The
resulting residue was dissolved in minimum amount of
hot H2O/THF (v/v, 1/1). This solution was cooled,
applied to a column packed with Dowex 50W-X8 resin,
and then eluted with H2O/THF (v/v, 1/1). Suitable
fractions were combined and concentrated in vacuo.
The white precipitate obtained was dissolved in hot
THF and the solution was cooled to rt and anhydrous
Et2O was added. The white precipitate was collected
and air dried to give 152 mg (82%) of the desired prod-
uct: mp 250±252 ꢀC; 1H NMR (DMSO-d6) d 1.02 (d,
2H), 2.40±2.70 (m, 2H), 2.75±3.15 (m, 2H), 4.03 (m,
1H), 4.07 (m, 1H), 4.54 (m, 1H), 4.86 (m, 1H), 4.96 (d,
1H), 7.05±7.30 (m, 5H), 6.90±7.40 (m, 4H), 7.54 (d, 1H),
7.40±7.9 (m, 5H), 8.12 (d, 1H), 8.61 (d, 1H); 13C NMR
(DMSO-d6) d 20.0, 36.6, 36.9, 50.4, 54.1, 58.3, 66.3,
126.0, 127.3, 127.8, 128.0, 129.1, 131.2, 133.7, 137.5,
166.1, 170.6, 170.8, 171.6, 171.8; MS (CI) m/e (rel.
intensity) 484 (MH+, 38), 219 (43), 122 (100); HRMS
calcd for C24H30N5O6 (MH+), 484.2196; found,
centrated in vacuo and the colorless syrup was triturated
with Et2O. The white precipitate was collected and
rinsed with Et2O and then dissolved in 2 mL dry DMF.
NMM (47 mL, 0.43 mmol) was added and the mixture
was cooled to 0 ꢀC. N-Boc-Asn (99 mg, 0.43 mmol),
.
EDC HCl (86 mg, 0.45 mmol), and HOBt (86 mg,
0.64 mmol) were dissolved in 2 mL dry THF at 0 ꢀC
under nitrogen. NMM (49 mL, 0.45 mmol) was added
and the mixture was stirred for 10 min at 0 ꢀC. The pre-
.
cooled solution of TFA Bpa-Thr-NH2 (0.43 mmol) and
NMM (47 mL, 0.43 mmol) in 2 mL dry DMF was added
and the reaction mixture was stirred at 0 ꢀC for 1 h and
at rt for 15 h. The reaction mixture was concentrated in
vacuo and the brown residue was triturated with 4 mL
5% citric acid. The precipitate collected was further tri-
turated with saturated NaHCO3 solution. The crude
product collected was crystallized from MeOH/H2O to
give 190 mg (76%) of a white solid: Rf 0.56 (MeOH/
EtOAc/Et3N, 3/3/1); mp 204±207 ꢀC; 1H NMR
(CD3OD) d 1.17 (d, 3H), 1.36 (s, 9H), 2.38 (m, 2H),
2.96±3.30 (m, 2H), 4.18 (m, 1H), 4.27 (m, 1H), 4.41 (m,
1H), 4.94 (m, 1H), 7.44±7.75 (m, 9H); 13C NMR
(DMSO-d6/CD3OD) d 20.1, 28.2, 37.3, 40.2, 51.4, 53.6,
58.4, 66.5, 78.3, 128.6, 129.5, 129.6, 129.7, 132.5, 135.0,
137.4, 143.2, 155.1, 170.6, 171.5, 171.8, 172.1, 195.6; MS
(FAB+) m/e (rel. intensity) 584 (MH+, 60), 484 (100),
224 (66), 79 (66); HRMS calcd for C29H38N5O8 (MH+),
584.2720; found, 584.2719.
.
484.2198. Anal. calcd for C24H29N5O6 0.5H2O: C, 58.53;
H, 6.14; N, 14.22. Found: C, 58.58; H, 6.40; N, 14.02.
N-Boc-Asn-Bpa-Thr-NH2 (8b). N-Boc-Bpa (6b, 296 mg,
.
0.80 mmol), EDC HCl (161 mg, 0.84 mmol), and HOBt
(162 mg, 1.20 mmol) were dissolved in 3 mL dry THF at
0 ꢀC while stirring. NMM (92 mL, 0.84 mmol) was added
and the mixture was stirred at 0 ꢀC under nitrogen for
N-Bz-Asn-Bpa-Thr-NH2 (3b). N-Boc-Asn-Bpa-Thr-NH2
(8b, 150 mg, 0.26 mmol) was dissolved in 5 mL TFA/
CH2Cl2 (v/v, 1/1) and the mixture was allowed to set at
rt for 30 min. The mixture was concentrated in vacuo
and the colorless oil was triturated with Et2O. The white
precipitate was collected by ®ltration and rinsed with
Et2O and then dissolved in 9 mL H2O/dioxane/DMF (v/
v/v, 1/1/1). Et3N (36 mL, 0.26 mmol) and Bz2O (64 mg,
0.28 mmol) were added and the mixture was stirred for
5 min. Et3N (20 mL, 0.14 mmol) was further added and
the reaction mixture was stirred at rt for 12 h. The mix-
ture was concentrated in vacuo and the white residue
was dissolved in warm H2O/THF (v/v, 1/1). Dowex
50W X8 resin was added to the cooled mixture and the
mixture was stirred slightly. The ®ltrate was con-
centrated in vacuo and the white residue was crystallized
from H2O/THF. The crude product was further recrys-
tallized from MeOH/THF to give 122 mg white solid,
81% yield: Rf 0.65 (MeOH/EtOAc, 1/1); mp 260±
261 ꢀC; 1H NMR (DMSO-d6/CD3OD) d 1.14 (d, 3H),
2.55±2.85 (m, 2H), 3.00±3.35 (m, 2H), 4.14 (m, 1H), 4.21
(m, 1H), 4.69 (m, 1H), 4.89 (m, 1H), 7.30±7.80 (m, 14H);
13C NMR (DMSO-d6/CD3OD) d 20.1, 36.5, 37.0, 50.5,
53.8, 58.5, 66.5, 127.5, 128.2, 128.5, 129.5, 131.5, 132.5,
133.8, 134.9, 137.3, 143.2, 166.3, 170.7, 171.1, 172.0,
172.1, 195.5; MS (FAB+) m/e (rel. intensity) 588 (MH+,
9), 233 (20), 157 (33), 105 (22), 79 (100); HRMS calcd
.
10 min. A precooled mixture of HCl Thr-NH2 (124 mg,
0.80 mmol) and NMM (88 mL, 0.80 mmol) in 3 mL dry
DMF was added and the mixture was stirred at 0 ꢀC for
1 h and at rt for 1 day. However, TLC still showed
unreacted starting material. Thirty-eight milligrams of
.
EDC HCl (0.20 mmol) and NMM (22 mL, 0.20 mmol)
was further added at 0 ꢀC and the reaction mixture was
stirred at rt for another day. The reaction mixture was
concentrated in vacuo and the yellowish residue was
partitioned between EtOAc and 5% citric acid solution.
The organic layer was separated and washed with satu-
rated NaHCO3 solution, dried, and concentrated in
vacuo. The resulting syrup was dissolved in minimum
amount of MeOH and hexane was slowly added. The
precipitate was collected and dried to give 277 mg (74%)
of N-Boc-Bpa-Thr-NH2 as a white solid: Rf 0.64
(MeOH/EtOAc/AcOH, 4/4/1); mp 143±145 ꢀC; 1H
NMR (CD3OD) d 1.17 (d, 3H), 1.30 (s, 9H), 2.96±3.31
(m, 2H), 4.23 (m, 1H), 4.28 (m, 1H), 4.43 (m, 1H), 7.44±
7.78 (m, 9H); 13C NMR (DMSO-d6/CD3OD) d 20.7,
29.1, 38.6, 57.4, 59.7, 68.1, 80.7, 129.8, 130.9, 131.1,
131.3, 133.9, 137.2, 139.1, 144.8, 157.6, 173.9, 174.6, 197.7.
N-Boc-Bpa-Thr-NH2 (200 mg, 0.43 mmol) was dissolved
in 6 mL TFA/CH2Cl2 (v/v, 1/1) and the mixture was
allowed to set at rt for 30 min. The mixture was con-

T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
1831
for C31H34N5O7 (MH+), 588.2458; found, 588.2459.
Anal. calcd for C31H33N5O7: C, 63.36; H, 5.66; N,
11.92. Found: C, 63.36; H, 5.75; N, 11.86.
was stirred at 0 ꢀC for 1 h and at rt for 1 day and then
concentrated in vacuo. The resulting residue was par-
titioned between EtOAc and 5% citric acid solution. The
organic layer was separated and washed with saturated
NaHCO3 solution and then concentrated in vacuo. The
resulting white residue was triturated with H2O/MeOH
(v/v, 2/1) at 0 ꢀC, collected, and dried to give 273 mg
(81%) of a white solid. The crude product was further
crystallized from MeOH/Et2O. Rf 0.58 (EtOAc/MeOH,
3/1); mp 196±198 ꢀC; 1H NMR (CD3OD/DMSO-d6) d
1.02 (d, 3H), 1.31 (s, 9H), 1.42 (m, 2H), 1.50 (m, 2H),
1.60±1.85 (m, 2H), 2.45±2.65 (m, 2H), 3.24 (t, 2H), 4.04
(m, 1H), 4.07 (m, 1H), 4.21 (m, 1H), 4.30±4.40 (covered
by HDO, 1H), 7.35±7.80 (m, 5H); 13C NMR (DMSO-
d6) d 20.1, 22.7, 28.2, 28.8, 31.5, 37.1, 39.1, 51.4, 53.1,
58.4, 66.4, 78.3, 127.2, 128.2, 131.0, 134.7, 155.1, 166.1,
171.7, 171.7, 171.9, 172.3.
Nꢀ-Boc-N"-Bz-L-lysine (9a). Ne-Bz-l-lysine (12, 1.0 g,
4.0 mmol) was dissolved in 20 mL NaOH solution
(0.20 M, 4.0 mmol) at 0 ꢀC and 20 mL dioxane was
added. (Boc)2O (960 mg, 4.4 mmol) was added and the
reaction mixture was stirred at rt overnight. The mixture
was adjusted by NaOH solution to pH 9 and was
washed with Et2O to remove unreacted (Boc)2O. The
aqueous layer was cooled to 0 ꢀC and 2 N HCl was
added slowly while stirring to adjust pH to 3. The acidic
aqueous layer was washed with EtOAc (3Â) and the
organic layers were combined, dried, and concentrated
in vacuo to give 1.35 g white foam in 96% yield: Rf 0.51
(EtOAc/MeOH, 1/1); 1H NMR (CDCl3/CD3OD) d
1.35±1.50 (m, 2H), 1.41 (s, 9H), 1.55±1.90 (m, 4H), 3.41
(t, 2H), 4.22 (m, 1H), 7.35±7.80 (m, 5H); 13C NMR
(CDCl3) d 22.5, 28.1, 28.6, 31.8, 39.7, 53.1, 79.7, 127.1,
128.3, 131.4, 134.0, 155.8, 168.5, 175.2; MS (FAB+) m/e
(rel intensity) 351 (MH+, 11), 295 (24), 251 (79), 105
(100); HRMS calcd for C18H27N2O5 (MH+), 351.1920;
found, 351.1902.
N-Bz-Asn-Lys(N"-Bz)-Thr-NH2 (4a). N-Boc-Asn-
Lys(Ne-Bz)-Thr-NH2 (10a, 169 mg, 0.30 mmol) was dis-
solved in 4 mL TFA/CH2Cl2 (v/v, 1/1) at rt and was
allowed to set for 30 min. The clear solution was con-
centrated in vacuo and the colorless oil was triturated
with Et2O. The white precipitate was collected, rinsed
with Et2O, dried, and then dissolved in 6 mL DMF/H2O
(v/v, 2/1) mixed solvent. The mixture was cooled to 0 ꢀC
and Et3N (42 mL, 0.30 mmol) was added followed by
Bz2O (71 mg, 0.33 mmol). The reaction mixture was
stirred for 5 min and Et3N (21 mL, 0.15 mmol) was fur-
ther added. The reaction mixture was stirred at rt over-
night and then concentrated in vacuo. The resulting
white residue was dissolved in 20 mL THF/H2O (v/v, 1/
1) and 5 mL bed volume of Dowex 50W-X8 resin (H+
form) was added and the mixture was gently stirred and
then ®ltered. The ®ltrate was concentrated in vacuo and
the resulting white residue was crystallized from THF/
H2O (v/v, 1/1) and then triturated with Et2O. The white
precipitate was collected, washed with Et2O, and dried
to give 138 mg (81%) of a white powder. Rf 0.40
(EtOAc/MeOH, 3/1); mp 266±268 ꢀC; 1H NMR
(CD3OD/DMSO-d6) d 0.99 (d, 3H), 1.32 (m, 2H), 1.47
(m, 2H), 1.55±1.80 (m, 2H), 2.62 (m, 2H), 3.19 (m, 2H),
3.99 (m, 1H), 4.03 (m, 1H), 4.22 (m, 1H), 4.74 (m, 1H),
7.35±7.85 (m, 5H); 13C NMR (DMSO-d6) d 20.1, 22.8,
28.9, 31.2, 37.0, 39.1, 50.8, 53.2, 58.2, 66.4, 127.2, 127.5,
128.3, 131.0, 131.5, 134.0, 134.7, 166.1, 166.4, 171.4,
171.6, 171.9, 172.2; MS (FAB+) m/e (rel intensity) 569
(MH+, 28), 105 (73), 79 (100); HRMS calcd for
C28H37N6O7 (MH+), 569.2724; found, 569.2725. Anal.
calcd for C28H36N6O7: C, 59.14; H, 6.38; N, 14.78.
Found: C, 59.31; H, 6.05; N, 14.63.
N-Boc-Asn-Lys(N"-Bz)-Thr-NH2 (10a). Na-Boc-Lys(Ne-
Bz)-OH (9a, 526 mg, 1.50 mmol), HOBt (304 mg,
.
2.25 mmol), and H-Thr-NH2 HCl (232 mg, 1.50 mmol)
were dissolved in 10 mL dry DMF and then cooled to
0 ꢀC while stirring. NMM (338 mL, 3.08 mmol) was
.
added followed by EDC HCl (303 mg, 1.58 mmol). The
reaction mixture was stirred at 0 ꢀC for 1 h and at rt for
18 h and then concentrated in vacuo. The resulting
brown residue was partitioned between 30 mL EtOAc
and 10 mL 5% citric acid solution. The organic layer
was further washed with saturated NaHCO3 solution,
dried, and concentrated in vacuo. The white residue
obtained was crystallized from EtOAc/MeOH to give
532 mg (79%) of a white solid: Rf 0.69 (EtOAc/MeOH,
1/1); mp 142±144 ꢀC; 1H NMR (CD3OD) d 1.08 (d, 3H),
1.37 (s, 9H), 1.42 (m, 2H), 1.59 (m, 2H), 1.60±1.85 (m,
2H), 3.32 (m, 2H), 3.98 (m, 1H), 4.22 (m, 2H), 7.35±7.80
(m, 5H); 13C NMR (CD3OD) d 20.4, 24.4, 28.9, 30.2,
32.3, 40.8, 56.9, 59.6, 68.0, 81.1, 128.4, 129.6, 132.7,
135.8, 158.5, 170.3, 175.2, 175.6; MS (FAB+) m/e (rel
intensity) 451 (MH+, 25), 351 (58), 188 (29), 105 (46),
79 (100); HRMS calcd for C22H35N4O6 (MH+),
451.2556; found, 451.2544.
N-Boc-Asn (139 mg, 0.60 mmol), HOBt (122 mg,
e
.
0.90 mmol), and H-Lys(N -Bz)-Thr-NH2 TFA (from
TFA-mediated Na-deprotection of 270 mg (0.60 mmol)
Na-Boc-(Ne-Bz)Lys-Thr-NH2) were dissolved in 5 mL
dry DMF and the mixture was cooled to 0 ꢀC while
stirring. NMM (135 mL, 1.23 mmol) was added followed
Nꢀ-Boc-N"-(4-Bz)Bz-L-Lysine (9b). Na-Boc-Ne-Cbz-l-
lysine-OTMSE (13, 1.76 g, 3.66 mmol) was dissolved in
12 mL MeOH and 120 mg Pd/C catalyst was added. The
reaction mixture was shaken in a Parr shaker under H2
.
by EDC HCl (121 mg, 0.63 mmol). The reaction mixture

1832
T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
.
.
pressure of 40 psi for 15 h. The mixture was ®ltered
through a pad of celite and the ®ltrate was concentrated
in vacuo to give 1.24 g (98% yield) N-Boc-l-Lysine 2-
trimethylsilylethyl ester as a clear oil. Rf 0.13 (EtOAc/
2.1 mmol), EDC HCl (211 mg, 1.1 mmol), and HCl Thr-
NH2 (155 mg, 1.0 mmol) were added. The reaction mix-
ture was stirred at 0 ꢀC for 1 h and at rt for 18 h and
then concentrated in vacuo. The resulting brown residue
was partitioned between EtOAc and 5% citric acid
solution. The organic layer was further washed with
saturated NaHCO3 solution, dried, and concentrated in
vacuo. The resulting white residue was crystallized from
MeOH/EtOAc to give 392 mg (71%) of Na-Boc-Lys[Ne-
(4-Bz)Bz]-Thr-NH2 as a white solid: Rf 0.64 (EtOAc/
MeOH, 3/1); mp 139±141 ꢀC; 1H NMR (CD3OD/
DMSO-d6) d 1.17 (d, 3H), 1.46 (s, 9H), 1.45±1.95 (m,
6H), 3.44 (t, 2H), 4.24 (m, 1H), 4.41 (m, 1H), 4.55 (m,
1H), 7.55±8.05 (m, 9H); 13C NMR (DMSO-d6) d 20.0,
23.1, 28.2, 28.8, 31.0, 55.0, 57.7, 66.3, 78.5, 127.4, 128.7,
129.5, 129.7, 133.0, 136.7, 138.1, 139.51 155.8, 165.5,
172.2, 172.3, 195.5; MS (FAB+) m/e (rel. intensity) 555
(MH+, 12), 455 (37), 209 (29), 155 (52), 119 (81), 85
(100); HRMS calcd for C29H38N4O7 (MH+), 555.2819;
found, 555.2795.
1
MeOH, 3/1); H NMR (CDCl3) d 0.03 (s, 9H), 0.98 (t,
2H), 1.42 (s, 9H), 1.50±1.95 (m, 6H), 2.79 (m, 2H), 4.19
(m, 3H), 5.19 (br, 1H), 5.36 (br, 2H); 13C NMR (CDCl3)
d -1.8, 17.0, 22.3, 28.0, 30.0, 31.8, 40.2, 53.1, 63.1, 79.1,
155.2, 172.5.
N-Boc-l-lysine-OTMSE (1.24 g, 3.58 mmol) was dis-
solved in 20 mL dry THF and 4-benzoylbenzoic acid
(810 mg, 3.58 mmol) and NMM (433 mL, 3.94 mmol)
were added. The mixture was cooled to 0 ꢀC and
.
EDC HCl (755 mg, 3.94 mmol) was added and the mix-
ture was stirred at 0 ꢀC for 1 h and at rt for 20 h. The
reaction mixture was concentrated in vacuo and the
resulting brown residue was partitioned between EtOAc
and 5% citric acid solution. The organic layer was fur-
ther washed with saturated NaHCO3 solution and
brine, dried by anhydrous MgSO4, and concentrated in
vacuo. A slightly yellowish oil (1.83 g) was obtained
N-Boc-Asn (128 mg, 0.55 mmol) and HOBt (111 mg,
0.83 mmol) were dissolved in 2 mL dry DMF and then
cooled to 0 ꢀC while stirring. EDC HCl (117 mg,
.
1
with 92% yield: Rf 0.68 (EtOAc/MeOH, 3/1); H NMR
(CDCl3) d 0.04 (s, 9H), 1.01 (t, 2H), 1.42 (s, 9H), 1.45±
2.00 (m, 6H), 3.49 (m, 2H), 4.22 (t, 2H), 4.27 (m, 1H),
5.14 (d, 1H), 6.49 (m, 1H), 7.45±7.90 (m, 9H); 13C NMR
(CDCl3) d -1.7, 17.1, 22.5, 28.1, 28.7, 31.8, 39.5, 53.3,
63.3, 79.3, 127.1, 128.2, 129.5, 129.6, 129.8, 132.7, 136.7,
137.8, 139.4, 155.5, 166.8, 172.7, 195.8.
0.61 mmol) and NMM (67 mL, 0.61 mmol) were added
and the mixture was stirred at 0 ꢀC under nitrogen for
5 min. A precooled solution of H-Lys[Ne-(4-Bz)Bz]-Thr-
a
.
NH2 TFA (from TFA-mediated N -deprotection of
305 mg (0.55 mmol) Na-Boc-Ne-(4-Bz)Bz-Lys-Thr-NH2)
and NMM (61 mL, 0.55 mmol) in 2.5 mL dry DMF was
added and the reaction mixture was stirred at 0 ꢀC for
1 h and at rt for 18 h. The mixture was concentrated in
vacuo and the brown residue was triturated with 4 mL
5% citric acid solution. The white precipitate was col-
lected and further triturated with saturated NaHCO3
solution. The white precipitate was collected by ®ltra-
tion and washed with water. The crude product was
crystallized from MeOH/H2O to give 315 mg white solid
with 86% yield: Rf 0.66 (EtOAc/MeOH, 1/1); mp 197±
199 ꢀC; 1H NMR (CD3OD/DMSO-d6) d 1.15 (d, 3H),
1.41 (s, 9H), 1.50 (m, 2H), 1.66 (m, 2H), 1.75±2.05 (m,
2H), 2.55±2.80 (m, 2H), 3.40 (t, 2H), 4.18 (m, 1H), 4.21
(m, 1H), 4.32 (m, 1H), 4.41 (m, 1H), 7.53±7.98 (m, 9H);
13C NMR (DMSO-d6) d 20.2, 22.9, 28.3, 29.0, 31.6,
37.3, 39.4, 51.5, 53.3, 58.6, 66.6, 78.7, 127.6, 128.9,
129.8, 130.0, 133.2, 137.0, 138.3, 139.4, 155.5, 165.8,
172.0, 172.1, 172.4, 172.7, 195.8; MS (FAB+) m/e (rel.
intensity) 669 (MH+, 16), 569 (28), 209 (34), 85 (32), 79
(100); HRMS calcd for C33H45N6O9 (MH+), 669.3248;
found, 669.3269.
Na-Boc-Ne-(4-Bz)Bz-l-lysine-OTMSE
(14,
1.80 g,
3.24 mmol) was dissolved in 4 mL THF and 1.0 M tetra-
butylammonium ¯uoride (9.7 mL, 9.7 mmol) was added
and the reaction mixture was stirred at rt overnight. The
mixture was concentrated in vacuo and then partitioned
between EtOAc and 5% citric acid solution. The
organic layer was then washed with saturated NaHCO3
solution and the resulting aqueous layer was cooled to
0 ꢀC, adjusted to pH 3 by cold saturated KHSO4 solu-
tion under vigorous stirring. The acidic aqueous layer
was washed with EtOAc (3Â) and the organic layers
were combined, dried, and concentrated in vacuo to give
1.32 g slightly yellowish oil with 90% yield: Rf 0.39
1
(EtOAc/MeOH, 3/1); H NMR (CDCl3) d 1.41 (s, 9H),
1.45±2.00 (m, 6H), 3.48 (m, 2H), 4.29 (m, 1H), 5.32 (d,
1H), 6.88 (m, 1H), 7.45±7.95 (m, 9H); 13C NMR
(CDCl3) d 19.4, 23.5, 28.1, 31.7, 39.8, 53.2, 60.3, 79.7,
127.2, 128.3, 129.7, 129.8, 132.8, 136.6, 137.5, 139.5,
155.9, 167.3, 175.4, 195.8; MS (FAB+) 455 (MH+, 4),
355 (74), 242 (100), 209 (76), 142 (36); HRMS calcd for
C25H31N2O6 (MH+), 455.2182; found, 455.2170.
N-Bz-Asn-Lys[N"-(4-Bz)Bz]-Thr-NH2 (4b). N-Boc-Asn-
Lys[Ne-(4-Bz)Bz]-Thr-NH2 (10b, 150 mg, 0.22 mmol)
was dissolved in 4 mL TFA/CH2Cl2 (v/v, 1/1) and was
allowed to set at rt for 30 min. The clear solution was
concentrated in vacuo and the resulting colorless oil was
N-Boc-Asn-Lys[N"-(4-Bz)Bz]-Thr-NH2 (10b). Na-Boc-
Lys[Ne-(4-Bz)Bz]-OH (9b, 455 mg, 1.0 mmol) and HOBt
(203 mg, 1.5 mmol) were dissolved in 8 mL dry DMF
and the mixture was cooled to 0 ꢀC. NMM (231 mL,

T. Xu et al./Bioorg. Med. Chem. 6 (1998) 1821±1834
1833
triturated with Et2O. The white precipitate was collected
and rinsed with Et2O and then dissolved in 6 mL DMF/
H2O (v/v, 2/1). Et3N (31 mL, 0.22 mmol) was added fol-
lowed by Bz2O (56 mg, 0.25 mmol). The reaction mix-
ture was stirred for 5 min and Et3N (15 mL, 0.11 mmol)
was further added and the mixture was stirred at rt
overnight. The reaction mixture was concentrated in
vacuo and the residue was triturated with Et2O. The
white precipitate was collected and redissolved in hot
THF/H2O (v/v, 1/1). The solution was cooled to rt and
5 mL bed volume of Dowex 50W-X8 (H+) resin was
added and the mixture was gently stirred. The mixture
was ®ltered and the ®ltrate was concentrated in vacuo.
The resulting white residue was crystallized from H2O/
THF (v/v, 1/1). 132 mg white solid was obtained, 88%
yield. The product was further triturated with Et2O to
give the pure tripeptide product. Rf 0.64 (EtOAc/
MeOH, 1/1); mp 244±246 ꢀC; 1H NMR (CD3OD/
DMSO-d6) d 1.03 (d, 3H), 1.39 (m, 2H), 1.53 (m, 2H),
1.60±1.80 (m, 2H), 2.65 (m, 2H), 3.27 (m, 2H), 4.05 (m,
the presence of the tested peptide by that observed in the
standard assay using Bz-Asn-Leu-Thr-NH2. Each assay
was run in duplicate and the result given (Table 1) is the
average of the duplicate assays. For each inhibitor
evaluated, the percentage inhibition was determined as
follows. Activity in either the absence or presence of an
inhibitor was obtained by determining the 3H (%) in the
aqueous phase due to formation of the glycopeptide
product.26
Ki determination of 3b and 4b. Each OST assay mixture
contained 200 mM chemically synthesized (GlcNAc)2-
PP-dolichol, 50 mM Tris, pH 7.5, 1% Triton X-100,
1 mM MnCl2, 90, 180, or 360 mM [14C]Bz-Asn-Leu-Thr-
NH2, 0, 18, 45, 90, 180, or 270 mM Bz-Asn-Bpa-Thr-
NH2 (3b) or Bz-Asn-[Ne-(4-Bz)Bz]Lys-Thr-NH2 (4b) in
DMSO (®nal [DMSO]=10% v/v), and 400 mg of P40
yeast microsome in a total volume of 100 mL. The assay
was worked up as described above and the aqueous
layer which contained the biosynthetic glycopeptide was
analyzed by reverse phase HPLC. A gradient pro®le of
10±25% MeOH in 20 min followed with 25% MeOH
isocratic condition was employed. The unreacted
[14C]Bz-Asn-Leu-Thr-NH2 (tR=69 min) and the glyco-
peptide product [14C]Bz-Asn(GlcNAc)2-Leu-Thr-NH2
(tR=76.5 min) were separated and quantitated using a
liquid scintillation counter. The rate of 14C-labeled glyco-
peptide formation was determined and the value of Ki
was obtained by a Dixon plot of these data (Fig. 2).
2H), 4.27 (m, 1H), 4.79 (m, 1H), 7.45±8.00 (m, 9H); 13
C
NMR (DMSO-d6) d 20.2, 22.9, 28.8, 31.3, 37.0, 39.3,
50.9, 53.3, 58.3, 66.5, 127.5, 127.6, 128.4, 128.8, 129.6,
129.8, 131.6, 133.1, 134.0, 136.8, 138.1, 139.1, 165.5,
166.5, 171.6, 171.8, 172.0, 172.3, 195.6; MS (FAB+) m/e
(rel. intensity) 673 (MH+, 14), 292 (17), 209 (30),
105 (60), 79 (100); HRMS calcd for C35H41N6O8
(MH+), 673.2986; found, 673.2991. Anal. calcd for
.
C35H40N6O8 0.5H2O: C, 61.66; H, 6.06; N, 12.33.
Found: C, 61.67; H, 6.42; N, 12.24.
Acknowledgements
Standard OST assay. The synthetic peptides 1±4 were
tested as OST substrates using either [3H]Dol-PP-OS or
[3H]Dol-PP-DS as previously described.7,26 The OST
assay procedure using [3H]Dol-PP-DS was carried out
as follows: The assay mixture contained ca. 6000 dpm
[3H]Dol-PP-DS, 50 mM Tris, pH 7.5, 1% Triton X-100,
1 mM MnCl2, 360 mM tested peptide in DMSO (®nal
[DMSO]=5% v/v), and 600 mg of P40 yeast microsome7
in a total volume of 100 mL. The assay mixture was
shaken at 250 rpm for 2 h at rt, then quenched by the
addition of 3 mL cold CHCl3/MeOH (v/v, 3/2). The
mixture was allowed to set on ice for 30 min and the
layers separated by centrifugation at 1000 g for 15 min.
The supernatant was removed and extracted with 1 mL
of 4 mM MgCl2. The biphasic mixture was thoroughly
agitated (Vortex) and then centrifuged at 1000 g for
another 15 min. The upper aq layer containing the water
This research has been supported by funds provided by
the Vahlteich Research Fund, the College of Pharmacy,
University of Michigan. We thank Mr. Xinggao Fang
for the synthesis of Dol-PP-DS, Dr. Barbara S. Gibbs
for the synthesis of [14C]Bz-Asn-Leu-Thr-NH2. We also
thank Ms. Carol Capelle for careful preparation of this
manuscript.
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