ALEWOOD ET AL.
(s, 2 H, OCH2), 2.35 and 2.30 (s, 3 H, CH3). 13C NMR (d6-DMSO) δ
170.1, 170.0, 158.3, 157.6, 154.7, 154.6, 138.2, 137.7, 134.4, 130.7,
130.6, 129.9, 128.9, 128.6, 128.3, 127.2, 126.0, 64.4, 20.9, 20.8. m/z
286 (MH+).
(R/S)-4-(tert-Butoxycarbonylamino-p-tolylmethyl)phenoxyacetic
acid 4b
This was prepared from 3b (16.2 g, 60 mmol) and di-tert-butyl
dicarbonate (13.5 g, 62 mmol) by the method for 4a giving white
prisms (13 g, 35 mmol, 58%) m.p. 170–171 ◦C.
1H NMR (300 MHz, d6-DMSO) δ 12.95 (broad s, 1 H), 7.83 (d,
J = 9.1 Hz, 1 H, NH), 7.22–7.07 (m, 6 H), 6.82 (AAꢁXXꢁ system
JAX + JAXꢁ = 8.6 Hz, 2 H), 5.71 (d, J = 9.2 Hz, 1 H, CH), 4.62 (s, 2 H,
OCH2), 2.25 (s, 3 H, CH3), 1.36 (s, 9 H, OtBu). 13C NMR (d6-DMSO) δ
170.2, 156.5, 155.1, 140.4, 135.9, 135.7, 128.8, 128.3, 127.0, 114.1,
78.0, 64.5, 56.9, 28.3, 20.6. m/z 255 (M-BocNH), 389 (MNH4+), 626
(M + 255), 643 (M + 272), 653, 743 (2 MH+), 760 (2 MNH4+).
(R/S)-4-(Amino-phenylmethyl)phenoxyacetic acid 3a
The oxime 2a (28.4 g, 105 mmol) was dissolved in boiling absolute
ethanol (300 ml) and stirred with a very substantial stirring bar
(or preferably mechanical stirring). Sodium (30 g) was added
through the condenser in portions as rapidly as possible, keeping
the reaction refluxing vigorously (about 30 min). Heating was
reapplied toward the end of the reaction and stirring was
continued until all of the sodium had dissolved. Cold water
(350 ml) was added and the mixture was stirred until all solids
had dissolved then the solution was acidified to pH 5 with conc.
HCl (about 120 ml). The mixture was left to crystallize overnight,
then the racemic product was filtered off, washed well with water
and dried giving a white powder (15.4 g, 56.7 mmol, 54%) m.p.
233–237 ◦C.
1H NMR (300 MHz, D2O + DCl + DSS) δ 7.55–7.30 (m, 7 H, ArH),
6.96 (AAꢁXXꢁ system JAX + JAXꢁ = 8.7 Hz, 2 H, ortho to O), 5.68 (s, 1
H, CHN), 4.72 (s, 2 H, OCH2). 13C NMR (D2O + DCl) δ 173.8, 158.1,
137.3, 130.8, 130.1, 129.8, 127.7, 116.0, 65.7, 58.4. λmax (KBr) cm−1
3200–2400, 1660–1470, 1423, 1338, 1302, 1253, 1200, 1056, 945,
851, 822, 696. m/z 258 (MH+), 241 (M-NH2).
Attachment of linker 4a/4b to Phe-PAM resin
The Boc-Phe-OCH2-PAM resin (0.5 mmol) was swollen in DMF
for 30 min then drained. The Boc group was removed using
neat TFA (2 × 1 min). The resin was flow washed with DMF for
1 min prior to neutralization with 10% DIEA in DMF (2 × 1 min).
The resin was then flow washed for 1 min with DMF. Linker 4a
(2 mmol) was dissolved in 3 ml DCM and a minimum amount
of DMF, then DIC (157 µl; 1 mmol) was added and the mixture
was activated at RT for ca 30 min (the formation of insoluble
N,N’-dicyclohexylurea was not observed). It was necessary to use
more DMF to dissolve linker 4b, but otherwise the procedure
was identical. The activated linker was then added to the drained
resin and the coupling efficiency measured after 60 min (>99.8%
incorporation) using the ninhydrin reaction [25]. The resin was
finally washed with DMF, DCM, then dried under nitrogen prior to
use in the syntheses.
(R/S)-4-(Amino-p-tolylmethyl)phenoxyacetic acid 3b
This was prepared from 2b (29.9 g, 105 mmol) using the procedure
for 3a yielding a white powder (17.0 g, 63 mmol, 60%) m.p.
235–238 ◦C.
Chain assembly Boc-chemistry
1H NMR (300 MHz, D2O + DCl + DSS) δ 7.35 (AAꢁXXꢁ system
The chain assembly of the peptides (0.25 mmol resin used) was
performed on a manual shaker system using HBTU activation and
in situ neutralization protocols [26] to couple the Boc-protected
amino acid to the resin. The Boc protecting group was removed
using 100% TFA, and DMF was used as both the coupling solvent
and for flowwashes throughout the cycle. The progress of the
assembly was monitored by the quantitative ninhydrin assay
[25].
Synthesis of CVID was performed on p-MBHA resin supplied
by Applied Biosystems and Bachem, as well as on synthesized
4a-, and 4b-Phe-PAM (PAM resin supplied by Applied Biosystems,
Bachem and Novabiochem) and 4a-Leu-PAM resin (PAM resins
supplied by Peptide International). The coupling efficiencies
were monitored and compared at a coupling time of 10 min
using the quantitative ninhydrin assay [25]. For results see
Figure 2.
JAX + JAXꢁ = 8.8 Hz, 2 H), 7.29 (m, 4 H), 7.01 (AAꢁXXꢁ system
JAX + JAXꢁ = 8.8 Hz, 2 H), 5.64 (s, 1 H), 4.77 (s, 2 H), 2.33 (s, 3 H). 13
C
NMR (D2O + DCl) δ 173.8, 158.1, 140.1, 134.5, 131.1, 130.6, 129.6,
127.7, 115.9, 65.6, 58.0, 21.0. m/z 255 (M-NH2), 543 (2 MH+).
(R/S)-4-(tert-Butoxycarbonylamino-phenylmethyl)phenoxyacetic
acid 4a
The amino acid 3a (14.6 g, 56.7 mmol) and K2CO3 (15.6 g) were
dissolved in water (120 ml) with heating, and then the solution
was cooled to room temperature. A solution of di-tert-butyl
dicarbonate (13.5 g, 62 mmol) in THF (120 ml) was added and the
mixture was stirred vigorously for 60 min. Water and ether were
added then the aqueous layer was acidified to pH 3 with citric acid
and extracted with EtOAc/DCM (2 : 1). The extracts were washed
with sat. brine, filtered through Celite (to remove a persistent
insoluble by-product), then dried (MgSO4) and evaporated to
dryness. The solid residue was recrystallized from ethanol giving
white prisms (10.62 g, 30 mmol, 53%) m.p. 154–156 ◦C.
αA-EIVA (1–14) was prepared from the linkers 4a and 4b linked
to a Phe-PAM resin. [Tyr(SO3H)15Tyr]-EpI was made with linker 4a
attached to Phe-PAM resin. [N11S]-PnIA was made on 4a-Phe-PAM
resin.
1H NMR (300 MHz, d6-DMSO) δ 7.89 (d, J = 9.3 Hz, 1 H, NH),
7.35–7.17 (m, 7 H, ArH), 6.34 (AAꢁXXꢁ system JAX + JAXꢁ = 8.7 Hz, 2
H, ortho to O), 5.76 (d, J = 9.3 Hz, 1 H, CHN), 4.63 (s, 2 H, OCH2),
1.39 (s, 9 H, (CH3)3. 13C NMR (d6-DMSO) δ 170.2, 156.6, 155.0, 143.3,
135.6, 128.3, 128.2, 127.0, 126.7, 114.1, 78.1, 64.4, 57.1, 28.3. λmax
(KBr) cm−1 3372, 3100–2500, 1744, 1683, 1611, 1506, 1446, 1391,
1365, 1303, 1252, 1223, 1170, 1082, 1046, 1022, 914, 882, 844, 805,
695. m/z 358 (MH+), 302 (M-tBu), 241 (M-BocNH).
Evaluation of stability of peptide-linker bond to TFA
The test peptide αA-EIVA assembled on both 4a- and 4b-Phe-PAM
resins was stirred at RT in TFA for 20 h. The TFA solution was
collected and diluted with water and lyophilized. Any resulting
solids were collected, weighed and examined by ESI-MS. The
amount of cleaved peptide product was compared to initial
peptide loading on the resin used.
c
wileyonlinelibrary.com/journal/psc Copyright ꢀ 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 551–557