Synthesis of orthogonally protected bis(aminomethyl)malonic acid, and its use as a key building block in the preparation of cyclic peptide conjugates of 2-N-alkyl-1,2,3,4-tetrahydroisoquinoline on a solid support

Article abstract of DOI:10.1002/1099-0690(200109)2001:18<3467::AID-EJOC3467>3.0.CO;2-A

Orthogonally protected bis(aminomethyl)malonic acid (1) was synthesized and used as a key building block for the construction of cyclic peptide conjugates on a solid support. The applicability of the building block was demonstrated by preparation of 19 ba

Full text of DOI:10.1002/1099-0690(200109)2001:18<3467::AID-EJOC3467>3.0.CO;2-A

FULL PAPER
Synthesis of Orthogonally Protected Bis(aminomethyl)malonic Acid, and Its
Use as a Key Building Block in the Preparation of Cyclic Peptide Conjugates
of 2-N-Alkyl-1,2,3,4-tetrahydroisoquinoline on a Solid Support
Pasi Virta,*[a] Jaana Rosenberg,[a] Tuomas Karskela,[a] Petri Heinonen,[a] and
Harri Lönnberg[a]
Keywords: Amino acids / Cyclic peptides / Peptide conjugates / Solid-phase synthesis
Orthogonally protected bis(aminomethyl)malonic acid (1)
preparation of 19 backbone cyclized/branched peptide con-
was synthesized and used as a key building block for the
jugates of N-2-alkyl-5-(3-aminopropoxy)-1,2,3,4-tetrahy-
construction of cyclic peptide conjugates on a solid support. droisoquinoline as stereoisomeric pairs.
The applicability of the building block was demonstrated by
Introduction
Cyclic peptides are conformationally less flexible than
their linear counterparts, which makes them useful tools for
probing the structure of receptor targets.[1] Conjugation of
small cyclic peptides to a core structure that is known to
Figure 1. Orthogonally protected bis(aminomethyl)malonic acid
(1).
exhibit affinity towards a given receptor may therefore pro-
vide useful sets of lead compounds for structural studies of
membrane receptors by chemical mapping. We have previ-
ously developed solid phase methods for the preparation of
both linear and branched β-peptide conjugates of N-2-al-
kyl-1,2,3,4-tetrahydroisoquinoline for such purposes,[2] us-
ing α,α-dialkylated and α,α-bisaminomethylated β-alanines
as the key building blocks. These conjugates were aimed at
probing the structure of the α2-adrenergic receptors. As an
extension of this approach we now report on the prepara-
tion of similar cyclic peptide conjugates on a solid support.
The methodology developed is based on utilization of an
orthogonally protected bis(aminomethyl)malonic acid (1) ,
bearing conventional Fmoc and Boc protecting groups on
the two amino functions, and with one of the carboxylic
acid groups protected as an allyl ester (Figure 1). The
Fmoc-protected amino group and the allyl-protected carb-
oxylic acid group have been exploited in construction of a
backbone-cyclized peptide moiety, while the Boc-protected
amino group and the free carboxylic acid group have been
used for insertion of this cyclic block into the linear peptide
chain. Thanks to the two dissimilar protecting groups on
the amino functions, 1 is chiral, and the peptide conjugates
are hence obtained as stereoisomeric pairs. As previously,[2]
5-(3-aminopropoxy)-1,2,3,4-tetrahydroisoquinoline immob-
ilized to hydroxymethyl polystyrene resin through a vinyl-
sulfonyl linker[3] was used as a handle for the peptide
chain assembly.
Results and Discussion
Synthesis of Bis(aminomethyl)malonic Acid (1)
The bis(aminomethyl)malonic acid building block (1) was
synthesized from benzylidene-protected pentaerythritol[4]
(2) as outlined in Scheme 1. 4-Methoxytritylation of one of
the unprotected hydroxy groups (3) was followed by dis-
placement of the other with phthalimide by a Mitsunobu
reaction (4),[5] removal of the phthaloyl group by hydrazin-
olysis (5), and protection of the deblocked amino group
with a Boc group (6). The next step, the opening of the 1,3-
dioxane ring by a palladium-catalyzed tert-butyl hydroper-
oxide oxidation,[6] was the key step of the synthesis. One of
the ring oxygens remained protected by a benzoyl group,
while the other was released as a hydroxy group (7) and was
subsequently displaced with phthalimide (8). Removal of
phthaloyl and benzoyl groups in a single step by hydrazino-
lysis was attempted, but this proved to yield an N-benzoyl-
ated product instead of a free amine. For this reason, the
benzoyl protecting group was removed by sodium methox-
ide-catalyzed transesterification (9) prior to hydrazinolysis
of the phthaloyl group (10). After protection of the amino
function with a Fmoc group (11), the hydroxy group was
oxidized to a carboxylic acid group (12) with PDC (pyridin-
ium dichromate) under neutral conditions.[7] The crude re-
action product was used for esterification of the carboxylic
acid function with allyl bromide 13, because 12 tended to
decompose during silica gel chromatography (several
[a]
Department of Chemistry, University of Turku,
20014 Turku, Finland
Fax: (internat) ϩ358-2/333-6700
E-mail: pasi.virta@utu.fi
Eur. J. Org. Chem. 2001, 3467Ϫ3473
WILEY-VCH Verlag GmbH, 69451 Weinheim, 2001
1434Ϫ193X/01/0918Ϫ3467 $ 17.50ϩ.50/0
3467

P. Virta, J. Rosenberg, T. Karskela, P. Heinonen, H. Lönnberg
FULL PAPER
Scheme 1. Synthesis of building block 1. (i) MMTrCl, Py, (ii) PhthNH, DEAD, Ph3P, THF, (iii) NH2NH2, OH2, EtOH, (iv) Boc2O,
NaOH, H2O, MeCN, (v) tBuOOH, Pd(OAc)2, benzene, (vi) NaOMe, MeOH, (vii) FmocCl, DIPEA, dioxane, (viii) PDC, DMF, (ix)
AllBr, DIPEA, DMF, (x) I2, MeOH, (xi) CrO3, H2SO4, acetone.
methods were tried). The MMTr group of 13 was removed were observed during standard piperidine treatment with a
with iodine in methanol,[8] and Jones oxidation of the re- reaction time of 15 min. Accordingly, unnecessarily basic
leased hydroxy function (14) accomplished the synthesis.
conditions were carefully avoided. Hydroxymethyl derivat-
ives of malonic acid have previously been reported to un-
dergo a similar transformation.[9] The removal of the Boc
group from 1 by TFA treatment did not, in turn, result in
any detectable side products; no elimination of the amino-
methyl group or hydrolysis of the allyl ester function could
be observed. Allyl protecting groups are frequently removed
by means of a tetrakis(triphenylphosphane)palladium(0)-
catalyzed reaction when a three-dimensional orthogonal
protecting strategy is applied.[10] This method could also be
used without difficulty to remove the allyl protection from
the carboxylic acid group of 1.
The Solid Support 15
Hydroxymethyl polystyrene derivatized with O-(vinylsul-
fonylethyl) groups was employed as the solid support. N-
(tert-butyloxycarbonyl)-protected
5-(3-aminopropoxy)-
1,2,3,4-tetrahydroisoquinoline was immobilized through its
N-2 atom by Michael addition onto the vinyl groups of this
resin, as described previously.[3] The relative quantity of the
tetrahydroisoquinoline compound was adjusted so that only
about one third of the vinylsulfonyl groups on the support
became derivatized. The final tetrahydroisoquinoline load-
ing was hence about 80 µmol gϪ1. The unchanged vinylsul-
fonyl groups were capped with methanol using DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene) as a base.
Synthesis of the Peptide Conjugates 16؊34
The 5-(3-aminopropoxy) side chain of the solid-sup-
ported tetrahydroisoquinoline core was first elongated with
one Nα-Fmoc-β-alanine unit, and 1 was then coupled to the
deprotected β-amino group using standard HATU [O-(7-
Removal of Protecting Groups on the Solid Support
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexa-
Conventional peptide synthesis strategies on a solid sup- fluorophosphate] procedures (Scheme 2.).[11] The Fmoc
port include removal of Fmoc and Boc groups with 20% protection was removed from the solid-supported 1, and a
piperidine in DMF and with 25% TFA in DCM (dichloro- short (three amino acids) peptide sequence was assembled
methane), respectively. The removal of the Fmoc protecting on the amino function. After this, the terminal Fmoc pro-
group from solid-supported 1 with piperidine proved to be tection and the allyl protection of 1 were removed, and the
rather readily accompanied by elimination of the unblocked cyclization (10 equiv. HATU, 20 equiv. DIPEA (N,N-diiso-
aminomethyl group, resulting in formation of a methyl- propylethylamine) in DMF, 15 h at room temperature) was
enemalonate derivative, but no traces of this side product performed. The Boc group was removed from 1, and coup-
3468
Eur. J. Org. Chem. 2001, 3467Ϫ3473

A Key Building Block for the Preparation of Cyclic Peptide Conjugates
FULL PAPER
ling with an appropriate Fmoc-protected amino acid (Gly,
Ala, and Phe) was carried out. Release from the support
was achieved by N-2 quaternization of the tetrahydroisoqui-
noline moiety with methyl iodide and subsequent triethyl-
amine treatment. Figure 2 shows an example of a crude reac-
tion product (stereoisomer pair 33).
Figure 2. HPLC chromatogram of the crude synthesis product of
conjugate 33 (refers to a pair of stereoisomers). Hypersil Hypurity
C18 (150ϫ4.6 mm, 5 µm), gradient from aqueous 0.1% TFA to
acetonitrile in 30 min, flow 1.0 mL minϪ1, detection at 276 nm.
Table 1. Required and found molecular weights (Mreq and [M ϩ
H]ϩfound) for the peptide conjugates synthesized (16؊34 cf. Scheme 2).
No.
P
AA
Mreq
[M ϩ H]ϩfound
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
GlyGlyβ-Ala
Glyβ-AlaGly
β-AlaGlyGly
β-Alaβ-AlaGly
β-AlaGlyβ-Ala
Glyβ-Alaβ-Ala
(β-Ala)3
β-AlaLeuβ-Ala
β-AlaLeuβ-Ala
β-AlaLeuβ-Ala
β-AlaPheβ-Ala
β-AlaPheβ-Ala
β-AlaPheβ-Ala
β-AlaGlnβ-Ala
β-AlaGlnβ-Ala
β-AlaGlnβ-Ala
β-AlaSerβ-Ala
β-AlaSerβ-Ala
β-AlaSerβ-Ala
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Gly
Ala
Phe
Gly
Ala
Phe
Gly
Ala
Phe
Gly
Ala
Phe
881.4
881.4
881.4
895.4
895.4
895.4
909.4
951.5
965.5
1041.5
985.5
999.5
1075.5
966.5
980.5
1056.5
925.4
939.4
1015.5
882.7
882.7
882.7
896.4
896.4
896.4
910.5
952.6
966.8
1042.9
986.8
1000.7
1076.5
967.3
981.5
1057.8
926.4
940.8
1016.8
Scheme 2. Synthesis of conjugates 16؊34. (i) TFA, DCM, (ii) pep-
tide assembly using HATU/Fmoc chemistry, (iii) (Ph3P)4Pd0,
DMSO, THF, 0.5  HCl, morpholine, (iv) piperidine, DMF, (v)
HATU, DIPEA, DMF, (vi) Fmoc-AA-OH, HATU, DIPEA, DMF,
(vii) MeI, DMF, (viii) Et3N, DCM.
Cyclization of peptides of fewer than seven α-amino acids
in length is usually difficult.[12] To evaluate the cyclization
efficiency, all the possible tripeptides consisting of glycine
and β-alanine were assembled on the amino group of 1 and
cyclized using a shorter reaction time (4 h) than in the final
library synthesis. It was clearly observed that the cyclization
efficiency correlated with the number of β-alanine units in
the peptide. When a Gly-Gly-Gly sequence was used, no
cyclization could be observed, even though this type of cyc-
lic backbone structure has been described previously.[12a] It
was also noticed that additional space between the tetrahy-
droisoquinoline moiety and 1 facilitates the cyclization.
This observation, in fact, prompted us to use one β-alanine
unit between 1 and the aminopropoxy tail of tetrahydroiso-
quinoline. Table 1 summarizes the peptide conjugates finally
prepared to demonstrate the applicability of the technique
developed. The authenticity of the products was verified by
HPLC/ESI-MS.
Experimental Section
General: NMR spectra were recorded on Bruker 200 NMR or
JEOL JNM-GX 400 spectrometers. The chemical shifts are given
in ppm from internal TMS. Ϫ The mass spectra were recorded on
7070E VG or PE SCIEX API 365 LC/ESI-MS/MS mass spectro-
meters. Ϫ RP HPLC analyses were performed using a Hypersil
150 ϫ 4.6 mm, 5 µm HypurityTM Elite C18 column, gradient from
aqueous 0.1% TFA to acetonitrile in 30 min, flow 1.0 mL minϪ1
detection at 276 nm.
,
Eur. J. Org. Chem. 2001, 3467Ϫ3473
3469

P. Virta, J. Rosenberg, T. Karskela, P. Heinonen, H. Lönnberg
44 mmol) was added to this solution. The reaction mixture was
FULL PAPER
Solid Support 15: The hydroxymethyl polystyrene support derivat-
ized with O-vinylsulfonylethyl groups[3] (1.0 g) was suspended in stirred at room temperature for 3 h, and the solvents were evapor-
DMF
(10 mL).
5-[3-(tert-Butyloxycarbonylamino)propoxy]-
ated off. The residue was dissolved in DCM (200 mL), and the
organic phase was washed with saturated aq. NaHCO3 and brine,
1,2,3,4-tetrahydroisoquinoline[2a] (40 mg, 0.13 mmol) in DMF
(2 mL) was added dropwise and the mixture was shaken at ambient and evaporated to dryness. The product was purified by silica gel
temperature for 20 h. The solid support was washed with DCM chromatography (30% EtOAc in petroleum ether), yielding 12.2 g
and methanol, and resuspended in a mixture of DCM (8.0 mL), (55%) of 3 as a white, solid foam. Ϫ 1H NMR (400 MHz, CDCl3):
methanol (4.0 mL), and DBU (0.5 mL). The suspension was shaken δ ϭ 2.17 (t, J ϭ 6.0 Hz, 1 H, OH), 3.45 (d, J ϭ 5.9 Hz, 2 H,
at ambient temperature for 20 h, washed with DCM and methanol, CH2OH), 3.62 (s, 2 H, CH2OMMTr), 3.78 (s, 3 H, OCH3), 3.79 (d,
and dried under reduced pressure. According to RP HPLC analysis
J ϭ 11.8 Hz, 2 H, 2 ϫ OCHHCq), 4.25 (d, J ϭ 11.8 Hz, 2 H, 2 ϫ
of the tetrahydroisoquinoline derivative released from the support, OCHHCq), 5.42 (s, 1 H, PhCH), 6.85 (m, 2 H, MMTr), 7.21Ϫ7.51
the loading was 80 µmol gϪ1
.
(m, 17 H, MMTr and Ph). Ϫ 13C NMR (100 MHz, CDCl3): δ ϭ
39.0 (Cq, pentaerythritol), 55.2 (OCH3), 63.7 (CH2OMMTr), 65.8
(CH2OH), 70.4 (OCH2Cq), 86.8 (Cq, MMTr), 102.2 (PhCH), 113.2
(MMTr), 126.3, 127.0, 128.0, 128.2, 128.2, 129.1, 130.4 (MMTr and
Ph), 135.2 (MMTr), 138.2 (Ph), 144.2, 158.7 (MMTr). Ϫ HRMS
(EI): Mϩ found 496.2236. C32H32O5 requires 496.2236.
Synthesis of Peptide Conjugates 16؊34: The Boc group was first
removed from support-bound 5-[3-(tert-butyloxycarbonylamino)p-
ropoxy]-1,2,3,4-tetrahydroisoquinoline (15 in Scheme 2) with the
aid of 25% TFA in DCM (1 h at room temperature). The fully
protected acyclic peptide chains containing 1 as the penultimate C-
terminal residue were then assembled using HATU/Fmoc chem-
istry (1 µmol scale). The support and (Ph3P)4Pd0 (5.8 mg, 5 mol.
equiv.) were then suspended in a mixture of DMSO, THF, aq. 0.5
 HCl, and morpholine (580 µL, 1:1:0.5:0.1, v/v/v/v), and the mix-
ture was shaken at room temperature for 45 min. This reaction time
was sufficient to complete the allyl deprotection. The support was
then filtered and washed successively with DMSO, DMF, 30%
Et3N in DMF, DMF, DCM, MeOH, and DMF, in that order. The
Fmoc group was removed with 20% piperidine in DMF (15 min at
room temperature), and the support was subjected to successive
washings with DMF, 30% Et3N in DMF, DMF, DCM, and MeOH.
The dried support was suspended in DMF (200 µL), and HATU
(3.8 mg, 10 molar equiv. in 180 µL DMF) and DIPEA (3.5 µL, 20
molar equiv. in 10 µL DMF) were added. The support was shaken
in this solution at room temperature for 15 h, and then filtered off,
washed with DMF, DCM, and MeOH, and dried. The Boc group
was removed by using 25% TFA in DCM (1 h at room temper-
ature), washed with DCM, 10% pyridine in DCM, DCM, and
MeOH, and dried. The appropriate amino acid (5 molar equiv. in
100 µL DMF), HATU (1.9 mg, 5 molar equiv. in 100 µL DMF),
and DIPEA (1.8 µL, 10 molar equiv. in 10 µL DMF) were added.
The mixture was shaken for 1 h at room temperature, and the solid
support was then filtered, washed with DMF, DCM, and MeOH,
and dried. The support was then shaken with a solution of methyl
iodide in DMF (MeI:DMF, 1:9, v/v) for 1 h, filtered off, washed
with DCM and MeOH, and dried. Triethylamine in DCM
(Et3N:DCM, 1:1, v/v) was added, and the mixture was shaken for
20 min. The solution was collected by filtration, and the solvents
evaporated off, producing 16؊34 as crude reaction products. Prior
to HPLC analysis, the residue was diluted with 0.1% aq. TFA. The
authenticity of the products was verified by HPLC/ESI-MS
(Table 1). The yield of each coupling was determined by assessing
the Fmoc released before addition of the next residue, by UV spec-
troscopy. On this basis the coupling efficiency was higher than 95%,
also when using 1 as a building block. In addition, the product was
in some cases (16, 22, 23, 28, 33) released from an aliquot of the
solid support after each coupling and analyzed by HPLC. Only
minor impurities gradually appeared. Accordingly, the overall yield
of the synthesis was approximately 50Ϫ70%. The chromatogram
depicted in Figure 2 illustrates the situation at the end of the syn-
thesis.
5-(4-Methoxytrityloxy)methyl-2-phenyl-5-phthalimidomethyl-1,3-
dioxane (4): Diethyl azodicarboxylate (DEAD; 7.6 mL, 48 mmol)
was added dropwise to a solution of 3 (12.1 g, 24 mmol), triphenyl-
phosphane (12.8 g, 48 mmol), and phthalimide (7.2 g, 48 mmol) in
THF (70 mL). The reaction mixture was stirred overnight at room
temperature, and evaporated to dryness. The crude reaction prod-
uct was purified by silica gel chromatography (30 to 50% EtOAc
in petroleum ether), yielding 15.0 g (98%) of 4 as a white, solid
foam. Ϫ 1H NMR (200 MHz, CDCl3): δ ϭ 3.58 (s, 2 H,
CH2NPhth), 3.65 (s, 2 H, CH2OMMTr), 3.78 (s, 3 H, OCH3), 3.82
(d, J ϭ 11.9 Hz, 2 H, 2 ϫ OCHHCq), 4.37 (d, J ϭ 11.9 Hz, 2 H,
2 ϫ OCHHCq), 5.35 (s, 1 H, PhCH), 6.81 (m, 2 H, MMTr),
7.17Ϫ7.53 (m, 17 H, MMTr and Ph), 7.69Ϫ7.87 (m, 4 H, Phth).
Ϫ
13C NMR (100 MHz, CDCl3): δ ϭ 39.8, 40.1 (CH2NPhth and
Cq, pentaerythritol), 55.1 (OCH3), 63.2 (CH2OMMTr), 70.6
(OCH2Cq), 86.4 (Cq, MMTr), 101.7 (PhCH), 113.0 (MMTr), 123.4
(Phth), 126.3, 126.7, 127.7, 128.1, 128.5, 129.0, 130.5 (MMTr and
Ph), 131.9, 134.1 (Phth), 135.4 (MMTr), 138.0 (Ph), 144.3, 158.4
(MMTr), 168.6 (CϭO, Phth). Ϫ HRMS (EI): Mϩ found 625.2461.
C40H35NO6 requires 625.2464.
5-Aminomethyl-5-(4-methoxytrityloxy)methyl-2-phenyl-1,3-dioxane
(5): Hydrazine monohydrate (3.7 mL, 76 mmol) was added to a
solution of 4 (12.1 g, 19 mmol) in 150 mL EtOH, and the reaction
mixture was stirred overnight at room temperature. The solution
was evaporated to dryness, dissolved in DCM (200 mL), filtered,
and washed with brine (200 mL). The organic phase was dried over
Na2SO4, and evaporated to dryness. The product was purified by
silica gel chromatography (5% MeOH, 0.5% Et3N in DCM),
yielding 5 (8.3 g, 81%) as a white, solid foam. Ϫ 1H NMR
(200 MHz, CDCl3): δ ϭ 2.59 (s, 2 H, CH2NH2), 3.51 (s, 2 H,
CH2OMMTr), 3.70 (d, J ϭ 11.2 Hz, 2 H, 2 ϫ OCHHCq), 3.78 (s,
3 H, OCH3), 4.24 (d, J ϭ 11.3 Hz, 2 H, 2 ϫ OCHHCq), 5.40 (s, 1
H, PhCH), 6.84 (m, 2 H, MMTr), 7.15Ϫ7.53 (m, 17 H, MMTr and
Ph). Ϫ 13C NMR (100 MHz, CDCl3): δ ϭ 38.5 (Cq, pentaerythri-
tol), 44.1 (CH2NH2), 55.1 (OCH3), 61.3 (CH2OMMTr), 71.6
(OCH2Cq), 86.1 (Cq, MMTr), 102.2 (PhCH), 113.1 (MMTr), 126.4,
126.8, 127.8, 128.2, 128.4, 129.0, 130.4 (MMTr and Ph), 135.6
(MMTr), 138.3 (Ph), 144.5, 158.5 (MMTr). Ϫ HRMS (EI): Mϩ
found 495.2411. C32H33NO4 requires 495.2410.
5-(tert-Butyloxycarbonylamino)methyl-5-(4-methoxytrityloxy)-
methyl-2-phenyl-1,3-dioxane (6): Compound 5 (7.3 g, 15 mmol) was
5-Hydroxymethyl-5-(4-methoxytrityloxy)methyl-2-phenyl-1,3- dissolved in 75% aq. acetonitrile (120 mL). Di-tert-butyl dicarbon-
dioxane (3): Benzylidene-protected pentaerythritol[4] (2) (15 g,
ate (3.5 g, 16 mmol) and 2  aqueous NaOH (9.5 mL, 19 mmol)
67 mmol) was coevaporated twice with dry pyridine, and dissolved were added, and the reaction mixture was stirred at room temper-
in the same solvent (60 mL). 4-Methoxytrityl chloride (13.7 g, ature overnight. DCM (300 mL) was added, and the organic phase
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Eur. J. Org. Chem. 2001, 3467Ϫ3473

A Key Building Block for the Preparation of Cyclic Peptide Conjugates
FULL PAPER
was washed with saturated NaHCO3, dried over Na2SO4, and evap-
orated to dryness. The crude product was purified by silica gel
chromatography (1% MeOH in DCM), yielding 6 (7.7 g, 88%) as
134.1 (Phth), 135.0 (MMTr), 144.0 (MMTr and Bz), 156.0 (CϭO,
Boc), 158.5 (MMTr), 166.1 (CϭO, Bz), 169.0 (CϭO, Phth). Ϫ
HRMS (EI): Mϩ found 740.3096. C45H44N2O8 requires 740.3098.
1
a white, solid foam. Ϫ H NMR (400 MHz, CDCl3): δ ϭ 1.39 [s,
2-(tert-Butyloxycarbonylamino)methyl-3-(4-methoxytrityloxy)-2-
phthalimidomethylpropan-1-ol (9): Compound 8 was dissolved in
sodium methoxide in methanol (1 , 100 mL), and the reaction
mixture was stirred at room temperature for 2 h. DCM (100 mL)
was added, and the mixture was directly loaded onto a silica gel
column. The column was eluted with DCM, yielding 9 (2.7 g, 61%)
9 H, (CH3)3], 3.00 (d, J ϭ 6.1 Hz, 2 H, CH2NHBoc), 3.51 (s, 2 H,
CH2OMMTr), 3.78 (d, J ϭ 11.7 Hz, 2 H, 2 ϫ OCHHCq), 3.79 (s,
3 H, OCH3), 4.17 (d, J ϭ 11.7 Hz, 2 H, 2 ϫ OCHHCq), 4.64 (t,
J ϭ 6.1 Hz, 1 H, NHBoc), 5.41 (s, 1 H, PhCH), 6.83 (m, 2 H,
MMTr), 7.21Ϫ7.54 (m, 17 H, MMTr and Ph). Ϫ 13C NMR
(50 MHz, CDCl3): δ ϭ 28.3 [(CH3)3], 38.5, 41.9 (CH2NHBoc and
Cq, pentaerythritol), 55.2 (OCH3), 61.4 (CH2OMMTr), 70.6
(OCH2Cq), 79.2 (Cq, Boc), 86.5 (Cq, MMTr), 102.2 (PhCH), 113.2
(MMTr), 126.3, 126.9, 127.8, 128.2, 128.3, 129.2, 130.3 (MMTr and
Ph), 135.4 (MMTr), 138.2 (Ph), 144.3, 158.6 (MMTr), 156.2 (Cϭ
O, Boc). Ϫ HRMS (EI): Mϩ found 595.2935. C37H41NO6 re-
quires 595.2934.
1
as a white, solid foam. Ϫ H NMR (400 Hz, CDCl3): δ ϭ 1.41 [s,
9 H, (CH3)3], 3.24 (s, 2 H, CH2OMMTr), 3.29Ϫ3.34 (m, 3 H,
CH2OH and CHHNHBoc), 3.40 (dd, J ϭ 12.4, J ϭ 7.0, 1 H,
CHHNHBoc), 3.62 (d, J ϭ 14.3 Hz, 1 H, CHHNPhth), 3.70 (d,
J ϭ 14.3 Hz, 1 H, CHHNPhth), 3.76 (s, 3 H, OCH3), 4.01 (m, 1
H, OH), 5.26 (m, 1 H, NHBoc), 6.76 (m, 2 H, MMTr), 7.14Ϫ7.33
(m, 12 H, MMTr), 7.70Ϫ7.89 (m, 4 H, Phth). Ϫ 13C NMR
(100 MHz, CDCl3): δ ϭ 28.3 [(CH3)3], 39.8, 41.1 (CH2NHBoc and
CH2NPhth), 46.3 (Cq, pentaerythritol), 55.1 (OCH3), 62.8
(CH2OMMTr), 64.9 (CH2OH), 79.6 (Cq, Boc), 86.6 (Cq, MMTr),
113.0 (MMTr), 123.4 (Phth), 126.8, 127.7, 128.4, 130.4 (MMTr),
132.0, 134.2 (Phth), 143.8 (MMTr), 157.3 (CϭO, Boc), 158.4
(MMTr), 169.2 (CϭO, Phth). Ϫ HRMS (EI): Mϩ found 636.2837.
C38H40N2O7 requires 636.2836.
2-Benzoyloxymethyl-2-(tert-butyloxycarbonylamino)methyl-3-(4-
methoxytrityloxy)propan-1-ol (7): A solution of tert-butyl hydroper-
oxide in benzene (15 mL, 6 , 90 mol) and a catalytic amount of
palladium acetate were added to a solution of 6 (7.6 g, 13 mmol)
in benzene (50 mL). The reaction mixture was stirred at room tem-
perature for 17 days, and concentrated by evaporation. The mixture
was dissolved in DCM (200 mL), washed with 10% aq. Na2SO3 (4
ϫ 150 mL) and brine (2 ϫ 200 mL), and evaporated to dryness.
The crude reaction product was purified by silica gel chromato-
graphy (30% EtOAc in petroleum ether), yielding 7 (5.8 g, 72%) as
3-Amino-2-(tert-butyloxycarbonylamino)methyl-2-(4-methoxytrityl-
oxy)methylpropan-1-ol (10): Compound 9 (5.2 g, 8.3 mmol) was dis-
solved in ethanol. Hydrazine monohydrate (2.0 mL, 42 mmol) was
added, and the reaction mixture was stirred at room temperature
overnight. Volatile components were removed by evaporation, and
the residue was purified by silica gel chromatography (0 to 10%
MeOH, 0.5% Et3N in CH2Cl2), yielding 10 (3.6 g, 86%) as a white,
solid foam. Ϫ 1H NMR (400 MHz, CDCl3): δ ϭ 1.39 [s, 9 H,
(CH3)3], 2.72 (s, 2 H, CH2NH2), 2.96 (s, 2 H, CH2OH), 3.11 (dd,
J ϭ 14.5, J ϭ 6.2, 1 H, CHHNHBoc), 3.33 (dd, J ϭ 14.5, J ϭ 7.4,
1 H, CHHNHBoc), 3.56 (d, J ϭ 11.6 Hz, 1 H, CHHOMMTr), 3.61
(d, J ϭ 11.6 Hz, 1 H, CHHOMMTr), 3.79 (s, 3 H, OCH3), 4.46 (m,
1 H, NHBoc), 7.42 (m, 2 H, MMTr), 7.21Ϫ7.33 (m, 8 H, MMTr),
7.40Ϫ7.43 (m, 4 H, MMTr). Ϫ 13C NMR (100 MHz, CDCl3): δ ϭ
28.3 [(CH3)3], 40.5 (CH2NHBoc), 44.2 (CH2NH2), 45.9 (Cq, penta-
erythritol), 55.2 (OCH3), 63.0 (CH2OMMTr), 65.4 (CH2OH), 79.5
(Cq, Boc), 86.3 (Cq, MMTr), 113.2, 127.0, 128.0, 128.2, 130.2,
135.0, 144.0 (MMTr), 157.2 (CϭO, Boc), 158.6 (MMTr). Ϫ HRMS
(ES): [M ϩ H]ϩ found 507.2869. C30H39N2O5 requires 507.2859.
1
a white, solid foam. Ϫ H NMR (200 MHz, CDCl3): δ ϭ 1.43 [s,
9 H, (CH3)3], 3.04 (d, J ϭ 9.0 Hz, 1 H, CHHOMMTr), 3.20 (m, 2
H, CHHOH and CHHNHBoc), 3.39 (d, J ϭ 9.0 Hz, 1 H,
CHHOMMTr), 3.54 (m, 2 H, CHHOH and CHHNHBoc), 3.69 (s,
3 H, OCH3), 4.08 (m, 1 H, OH), 4.40 (d, J ϭ 11.0 Hz, 1 H,
CHHOBz), 4.54 (d, J ϭ 11.0 Hz, 1 H, CHHOBz), 4.88 (m, 1 H,
NH), 6.78 (m, 2 H, MMTr), 7.20Ϫ7.70 (m, 15 H, MMTr and Bz),
7.93 (m, 2 H, Bz). Ϫ 13C NMR (50 MHz, CDCl3): δ ϭ 28.0
[(CH3)3], 39.7 (CH2NHBoc), 45.4 (Cq, pentaerythritol), 54.7
(OCH3), 60.7, 60.8 (CH2OMMTr and CH2OH), 63.4 (CH2OBz),
79.6 (Cq, Boc), 86.0 (Cq, MMTr), 112.9 (MMTr), 126.6, 127.6,
128.0, 129.0, 129.4, 129.6, 130.1 (MMTr and Bz), 132.8 (Bz), 134.8
(MMTr), 143.9, 144.1 (MMTr and Bz), 157.3 (CϭO, Boc), 158.3
(MMTr), 166.2 (CϭO, Bz). Ϫ HRMS (EI): Mϩ found 611.2881.
C37H41NO7 requires 611.2883.
2-Benzoyloxymethyl-N-(tert-Butyloxycarbonyl)-2-(4-methoxytrityl-
oxy)methyl-NЈ-phthaloylpropane-1,3-diamine (8): DEAD (2.9 mL, 2-(tert-Butyloxycarbonylamino)methyl-2-(9-fluorenylmethoxy-
19 mmol) was added dropwise to a solution of 7 (9.4 g, 15 mmol), carbonylamino)methyl-3-(4-methoxytrityloxy)propan-1-ol (11): 9-
triphenylphosphane (4.4 g, 17 mmol), and phthalimide (2.5 g, Fluorenylmethoxycarbonyl chloride (1.9 g, 7.3 mmol) was added
17 mmol) in THF (100 mL). The reaction mixture was stirred at portionwise to a solution of 10 (3.7 g, 7.2 mmol) and DIPEA (1.3
room temperature for 2 days, with addition of reagents or refluxing
sometimes being necessary to complete the reaction. The reaction stirred overnight at room temperature and evaporated to dryness.
solvents were evaporated to dryness, and the residue was purified The product was purified by silica gel chromatography (DCM),
by silica gel chromatography (30 to 50% EtOAc in petroleum yielding 11 (4.8 g, 91%) as a white, solid foam. Ϫ 1H NMR
ether), yielding 11.0 g (96%) of 8 as a white solid foam. Ϫ 1H NMR
(200 MHz, CDCl3): δ ϭ 1.42 [s, 9 H, (CH3)3], 2.60 (dd, J ϭ 14.3,
mL, 7.2 mmol) in dioxane (40 mL). The reaction mixture was
(200 MHz, CDCl3): δ ϭ 1.39 [s, 9 H, (CH3)3], 3.13Ϫ3.42 (m, 4 H, J ϭ 5.3, 1 H, CHHNHBoc), 2.77 (dd, J ϭ 14.3, J ϭ 5.3, 1 H,
CH2NHBoc and CH2OMMTr), 3.71 (d, J ϭ 14.4 Hz, 1 H, CHHNHBoc), 2.93 (d, J ϭ 9.1 Hz, 1 H, CHHOMMTr), 3.10 (d,
CHHNPhth), 3.71 (s, 3 H, OCH3), 4.05 (d, J ϭ 14.4 Hz, 1 H, J ϭ 9.1 Hz, 1 H, CHHOMMTr), 3.17 (m, 2 H, CH2NHFmoc),
CHHNPhth), 4.45 (d, J ϭ 11.6 Hz, 1 H, CHHOBz), 4.59 (d, J ϭ
3.40 (m, 2 H, CH2OH), 3.74 (s, 3 H, OCH3), 4.04 (m, 1 H, OH),
11.6 Hz, 1 H, CHHOBz), 5.61 (t, J ϭ 6.5 Hz, 1 H, NHBoc), 6.74 4.17 (m, 1 H, OCH2CH, Fmoc), 4.34 (m, 2 H, OCH2CH, Fmoc),
(m, 2 H, MMTr), 7.12Ϫ7.50 (m, 15 H, MMTr and Bz), 7.62Ϫ7.88 4.97 (m, 2 H, NHFmoc and NHBoc), 6.86 (m, 2 H, MMTr),
(m, 6 H, Phth and Bz). Ϫ 13C NMR (50 MHz, CDCl3): δ ϭ 28.3
7.16Ϫ7.52 (m, 18 H, MMTr and Fmoc), 7.78 (m, 2 H, Fmoc). Ϫ
[(CH3)3], 39.6, 41.9 (CH2NPhth, CH2NHBoc), 45.3 (Cq, pentaer- 13C NMR (50 MHz, CDCl3): δ ϭ 28.3 [(CH3)3], 39.3, 40.2
ythritol), 55.0 (OCH3), 63.4 (CH2OMMTr), 65.7 (CH2OBz), 79.0 (CH2NHBoc and CH2NHFmoc), 46.1 (Cq, pentaerythritol), 47.2
(Cq, Boc), 86.6 (Cq, MMTr), 113.1 (MMTr), 123.4 (Phth), 126.8,
127.8, 128.2, 128.4, 129.6, 130.4, 131.7 (MMTr and Bz), 132.8,
(OCH2CH, Fmoc), 55.2 (OCH3), 61.3 (CH2OH), 66.9
(CH2OMMTr), 67.1 (OCH2CH, Fmoc), 79.7 (Cq, Boc), 86.3 (Cq,
Eur. J. Org. Chem. 2001, 3467Ϫ3473
3471

P. Virta, J. Rosenberg, T. Karskela, P. Heinonen, H. Lönnberg
FULL PAPER
MMTr), 113.3 (MMTr), 120.0, 125.0 (Fmoc), 127.1, 127.7, 128.0, was evaporated to dryness, and the residue was dissolved in DCM
128.3 (MMTr and Fmoc), 130.1, 135.3 (MMTr), 141.3 (Fmoc), (200 mL), and washed several times with neutral aq. Na2SO3 (10%,
143.9, 144.1 (MMTr and Fmoc)157.8, 157.8 (CϭO, Fmoc and
m/v). The organic phase was dried over Na2SO4, and evaporated
Boc), 158.6 (MMTr). Ϫ HRMS (ES): [M ϩ H]ϩ found 729.3528. to dryness. The product was purified by silica gel chromatography
C45H49N2O7 requires 729.3540.
(20 to 50% EtOAc in petroleum ether), yielding 14 (1.2 g, 63%) as
a white, solid foam. Ϫ H NMR (200 MHz, CDCl3): δ ϭ 1.45 [s,
1
2-(tert-Butyloxycarbonylamino)methyl-2-(9-fluorenylmethoxy-
carbonylamino)methyl-3-(4-methoxytrityloxy)propanoic Acid (12):
Compound 11 (0.65 g, 0.89 mmol) and PDC (3.4 g, 9 mmol) were
dissolved in DMF (10 mL) and the reaction mixture was stirred
overnight at room temperature. Water (100 mL) was added, and
the organic components were extracted into diethyl ether (4 ϫ
20 mL). The organic phase was dried over NaSO4, and evaporated
to dryness. The resulting oil was purified by silica gel chromato-
graphy (0 to 20% MeOH in DCM), yielding 12 (82 mg, 12%) as a
colorless oil. The reason for this low yield was that 12 partially
decomposed on silica gel. Ϫ 1H NMR (200 MHz, CDCl3): δ ϭ
1.39 [s, 9 H, (CH3)3], 3.25Ϫ3.45 (m, 4 H, CH2NHBoc and
CH2OMMTr), 3.45Ϫ3.60 (m, 2 H, CH2NHFmoc), 3.71 (s, 3 H,
OCH3), 4.14 (t, J ϭ 6.0 Hz, 1 H, OCH2CH, Fmoc), 4.26 (d, J ϭ
6.0 Hz, 2 H, OCH2CH, Fmoc), 5.12, 5.62 (m, 1H and m, 1 H,
NHFmoc and NHBoc) 6.77 (m, 2 H, MMTr), 7.10Ϫ7.56 (m, 18
H, MMTr and Fmoc) 7.75 (m, 2 H, Fmoc). Ϫ 13C NMR (50 MHz,
9 H, (CH3)3], 3.09 (dd, J ϭ 14.6, J ϭ 5.6, 1 H, CHHNHBoc), 3.19
(dd, J ϭ 14.6, J ϭ 5.4, 1 H, CHHNHBoc), 3.55 (d, J ϭ 7.8 Hz, 2
H, CH2OH), 3.59 (m, 2 H, CH2NHFmoc), 4.25 (m, 2 H, OH and
OCH2CH, Fmoc), 4.42 (m, 2 H, OCH2CH, Fmoc), 4.65 (m, 2 H,
OCH2CHϭCH2), 5.22Ϫ5.39 (m, 2 H, OCH2CHϭCH2), 5.54 (m, 2
H, NHFmoc and NHBoc), 5.90 (m, 1 H, OCH2CHϭCH2)
7.26Ϫ7.44 (m, 4 H, Fmoc), 7.58 (m, 2 H, Fmoc), 7.77 (m, 2 H,
Fmoc). Ϫ 13C NMR (50 MHz, CDCl3): δ ϭ 28.3 [(CH3)3], 39.4,
40.3 (CH2NHFmoc and CH2NHBoc), 47.2 (OCH2CH, Fmoc),
55.0 (Cq, pentaerythritol), 62.4 (CH2OH), 65.7 (OCH2CHϭCH2),
67.1 (OCH2CH, Fmoc), 80.3 (Cq, Boc), 118.6 (OCH2CHϭCH2),
120.0, 125.0, 127.1, 127.8 (Fmoc), 131.7 (OCH2CHϭCH2), 141.3,
143.7 (Fmoc), 157.6, 157.8 (CϭO, Boc and Fmoc), 172.8 (CO-
OAll). Ϫ HRMS (ES): [M ϩ Na]ϩ found 533.2253. C28H34N2O7Na
requires 533.2264.
2-(tert-Butyloxycarbonylamino)methyl-2-(9-fluorenylmethoxy-
carbonylamino)methylmalonic Acid Monoallyl Ester (1): A solution
of CrO3 (0.34 g, 3.4 mmol) and H2SO4 (0.34 mL) in the minimum
possible amount of water was added dropwise to a solution of 14
(1.2 g, 2.3 mmol) in acetone (40 mL). The reaction mixture was
stirred at room temperature for 4 hours. The reaction was quenched
using 2-propanol, and the mixture concentrated by evaporation.
Chloroform was added (150 mL), and the organic phase was
washed with water (2 ϫ 30 mL) and brine (2 ϫ 30 mL), dried over
Na2SO4, and evaporated to dryness. Compound 14 was obtained
as a white, solid foam in an almost quantitative yield (1.2 g, 97%).
CDCl3):
δ ϭ 28.3 [(CH3)3], 40.5, 41.0 (CH2NHBoc and
CH2NHFmoc), 47.1 (OCH2CH, Fmoc), 55.1 (Cq, pentaerythritol),
55.1 (CH2OMMTr), 63.8 (CH2OMMTr), 66.9 (OCH2CH, Fmoc),
79.5 (Cq, Boc), 86.5 (Cq, MMTr), 113.2 (MMTr), 119.9, 125.2
(Fmoc), 127.0, 127.6, 127.8, 128.1, 128.3 (MMTr and Fmoc), 130.2,
135.1 (MMTr), 141.2 (Fmoc), 143.9 (MMTr and Fmoc), 156.6,
156.9 (CϭO, Boc and Fmoc), 158.5 (MMTr), 176.0 (COOH). Ϫ
HRMS (ES): [M ϩ K]ϩ found 781.2882. C45H46N2O8K requires
781.2891.
Allyl 2-(tert-Butyloxycarbonylamino)methyl-2-(9-fluorenylmethoxy-
Ϫ
1H NMR (200 MHz, CDCl3): δ ϭ 1.44 [s, 9 H, (CH3)3],
carbonylamino)methyl-3-(4-methoxytrityloxy)propanoate
(13):
3.70Ϫ3.79 (m, 4 H, CH2NHBoc and CH2NHFmoc), 4.22 (m, 1 H,
OCH2CH, Fmoc), 4.34 (m, 2 H, OCH2CH, Fmoc), 4.63 (m, 2 H,
OCH2CHϭCH2), 5.18Ϫ5.34 (m, 2 H, OCH2CHϭCH2), 5.34, 6.02
(m, 1 H and m, 1 H, NHBoc and NHFmoc), 5.89 (m, 1 H,
OCH2CHϭCH2), 7.26Ϫ7.42 (m, 4 H, Fmoc), 7.58 (m, 2 H, Fmoc),
7.75 (m, 2 H, Fmoc). Ϫ 13C NMR (50 MHz, CDCl3): δ ϭ 28.5
[(CH3)3], 41.2, 41.6 (CH2NHBoc and CH2NHFmoc), 47.1
(OCH2CH, Fmoc), 59.1 (Cq, malonic acid), 66.7 (OCH2CHϭ
CH2), 67.3 (OCH2CH, Fmoc), 80.4 (Cq, Boc), 119.0 (OCH2CHϭ
CH2), 120.0, 125.2, 127.1, 127.8 (Fmoc), 131.3 (OCH2CHϭCH2),
141.3, 143.8 (Fmoc), 128.5, 128.9 (CϭO, Boc and Fmoc), 168.6
(COOH), 171.6 (COOAll). Ϫ HRMS (ES): [M ϩ Na] found
547.2059. C28H32N2O8Na requires 547.2056.
Crude 12 (from 4.8 g/6.6 mmol of 11, not purified by silica gel chro-
matography) was dissolved in DMF (10 mL). Freshly distilled allyl
bromide (2.8 mL, 33 mmol) and DIPEA (0.55 mL, 3.3 mmol) were
added. The reaction mixture was stirred for 50 hours at room tem-
perature, and evaporated to dryness. The product was purified by
silica gel chromatography (0 to 1.5% MeOH in DCM), yielding 13
(2.9 g, 56% from 11) as a white, solid foam. Ϫ 1H NMR (200 MHz,
CDCl3): δ ϭ 1.39 [s, 9 H, (CH3)3], 3.20 (dd, J ϭ 14.7, J ϭ 7.1, 1
H, CHHNHBoc), 3.31 (m, 3 H, CHHNHBoc and CH2OMMTr),
3.49 (dd, J ϭ 14.3, J ϭ 6.7, 1 H, CHHNHFmoc), 3.56 (dd, J ϭ
14.3, J ϭ 6.5, 1 H, CHHNHFmoc), 3.74 (s, 3 H, OCH3), 4.15 (m,
1 H, OCH2CH, Fmoc), 4.28 (m, 2 H, OCH2CH, Fmoc), 4.69 (m,
2 H, OCH2CHϭCH2), 5.03 (m, NHFmoc), 5.41Ϫ5.23 (m, 2 H,
OCH2CHϭCH2), 5.48 (m, 1 H, NHBoc), 5.98 (m, 1 H, OCH2CHϭ
CH2) 6.81 (m, 2 H, MMTr), 7.58Ϫ7.11 (m, 18 H, MMTr and
Fmoc), 7.76 (m, 2 H, Fmoc). Ϫ 13C NMR (50 MHz, CDCl3): δ ϭ
28.3 [(CH3)3], 36.2, 37.0 (CH2NHBoc and CH2NHFmoc), 47.2
(OCH2CH, Fmoc), 53.4 (Cq, pentaerythritol), 55.1 (OCH3), 66.0
(OCH2CHϭCH2), 66.9 (OCH2CH, Fmoc), 79.4 (Cq, Boc), 86.3
(Cq, MMTr), 113.2 (MMTr), 118.8 (OCH2CHϭCH2), 119.9, 125.2
(Fmoc), 127.0, 127.7, 127.9, 128.3 (MMTr and Fmoc), 130.2
(MMTr), 132.0 (OCH2CHϭCH2), 135.0 (MMTr), 141.3 (Fmoc),
143.9 (MMTr and Fmoc), 156.5, 156.9 (CϭO, Boc and Fmoc),
158.6 (MMTr), 173.7 (COOAll). Ϫ HRMS (ES): [M ϩ Na]ϩ found
805.3516. C48H50N2O8Na requires 805.3465.
Acknowledgments
The authors wish to thank Dr. Mikko Ora for all those hours with
LC/ESI-MS/MS triple quadrupole mass spectrometer.
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