Synthesis and solid-phase application of suitably protected γ-hydroxyvaline building blocks

Article abstract of DOI:10.1021/jo070436y

(Chemical Equation Presented) Recently, an unexpected modified residue, γ-hydroxy-D-valine (D-Hyv), was identified within ribosomally expressed polypeptide chains of four conopeptides from the venoms of Conus gladiator and Conus mus. To assemble Hyv-conta

Full text of DOI:10.1021/jo070436y

Synthesis and Solid-Phase Application of Suitably Protected
γ-Hydroxyvaline Building Blocks
Mare Cudic, Frank Mar´ı, and Gregg B. Fields*
Department of Chemistry & Biochemistry, Florida Atlantic UniVersity, Boca Raton, Florida 33431
fieldsg@fau.edu
ReceiVed March 2, 2007
Recently, an unexpected modified residue, γ-hydroxy-D-valine (D-Hyv), was identified within ribosomally
expressed polypeptide chains of four conopeptides from the venoms of Conus gladiator and Conus mus.
To assemble Hyv-containing peptides, we have explored several routes for the synthesis of appropriately
functionalized Hyv building blocks. D-Hyv was produced from D-Val by using a variation of the previously
published K2PtCl4/CuCl2 oxidative method. Direct synthesis of Boc- or Cbz-D-Hyv lactone proceeded in
low yield; additionally, the lactones are too unreative for solid-phase applications. 9-Borabicyclononane
or copper-complexed D-Hyv was prepared and treated with tert-butyldimethylsilyl trifluoromethanesulfonate
(TBDMSOTf) to produce D-Hyv(O-TBDMS). The most efficient complex disruption was achieved by
Chelex 110 resin (Na+ form) treatment of copper-complexed D-Hyv(O-TBDMS). Reaction of D-Hyv-
(O-TBDMS) with Fmoc-OSu produced Fmoc-D-Hyv(O-TBDMS) in 26% yield from D-Val. The Fmoc-
D-Hyv(O-TBDMS) diastereomers were separated by preparative RP-HPLC in 13% yield from D-Val.
Fmoc-D-Hyv(O-TBDMS) was used for the synthesis of the conopeptide gld-V* from Conus gladiator.
The isolated synthetic and natural products had coincidental mass and NMR spectra. The methodology
presented herein will greatly facilitate biological studies of Hyv-containing sequences, such as receptor
responses to hydroxylated versus nonhydroxylated conopeptides and the relative susceptibility of proteins
to modification by oxidative stress.
Introduction
lation of Arg provides trypsin resistance to these mussel glue
proteins. Recently, an unexpected modified residue, γ-hydroxy-
D-valine (D-Hyv), was identified within ribosomally expressed
polypeptide chains of four conopeptides from the venoms of
Conus gladiator and Conus mus.3 These conopeptides were the
first known examples of a naturally occurring polypeptide chain
containing Hyv. In general, γ-hydroxyamino acids are not that
common in nature (except γ-hydroxy-Pro) since a hydroxyl
group in the γ-position can undergo intramolecular cyclization
to form a lactone, cleaving the peptide bond. For example,
N-acetyl-γ-L-hydroxyvaline lactone had been isolated from
streptomycete obtained from marine sediments.4 The stability
of Hyv within conopeptides has been explained by the D-
Hydroxylated amino acids constitute an important modifica-
tion in proteins. The best characterized of the post-translationally
hydroxylated amino acids are γ-hydroxy-Pro (Hyp) and δ-hy-
droxy-Lys (Hyl), which are commonly found in collagen.
γ-Hydroxy-L-Pro offers enhanced stabilization of the collagen
triple-helix compared with L-Pro, whereas δ-hydroxy-L-Lys is
the primary site of collagen glycosylation and participates in
intermolecular collagen cross-links.1 γ-Hydroxy-Arg has been
found as part of the sequence of polyphenolic proteins that form
the adhesive plaques of marine mussel species.2 The hydroxy-
* Address correspondence to this author. Phone: 561-297-2093. Fax: 561-
297-2759.
(1) Kivirikko, K. I.; Myllyla¨, R.; Pihlajaniemi, T. In Post-translational
Modifications of Proteins; Harding, J. J., Crabbe, M. J. C., Eds.; CRC
Press: Boca Raton, FL 1992; pp 1-51.
(2) Papov, V. V.; Diamond, T. V.; Biemann, K.; Waite, J. H. J. Biol.
Chem. 1995, 270, 20183-20192.
(3) Pisarewicz, K.; Mora, D.; Pflueger, F. C.; Fields, G. B.; Mar´ı, F. J.
Am. Chem. Soc. 2005, 127, 6207-6215.
(4) Hernandez, I. L. C.; Godinho, M. J. L.; Magalhaes, A.; Schefer, A.
B.; Ferreira, A. G.; Berlinck, R. G. S. J. Nat. Prod. 2000, 63, 664-
665.
10.1021/jo070436y CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/21/2007
J. Org. Chem. 2007, 72, 5581-5586
5581

Cudic et al.
SCHEME 1. Synthesis of Boc- or Cbz-Hyv
configuration at the R-carbon in conjunction with specific
interactions with the surrounding L-amino acids.3
Conopeptides elicit a wide range of strong neurophysiological
responses by targeting specific ion channels and receptors with
very high affinities and selectivities.5 As such they are promising
therapeutic agents. Given this role, there is much interest in
developing efficient methods for conopeptide synthesis.6 Ad-
ditionally, Hyv may be a useful marker for studying protein
oxidation in biological systems under oxidative stress.7 We have
presently considered the synthesis of Hyv derivative suitably
protected for Fmoc solid-phase chemistry and its subsequent
incorporation into a conopeptide sequence. The synthesis of Hyv
derivatives is complicated by the fact that Hyv contains two
asymmetric centers and therefore exists as four stereoisomers:
L-iso [(S,S)], D-iso [(R,R)], L-allo [(S,R)], and D-allo [(R,S)]. Hyv
has been prepared previously as a racemic mixture by a modified
Erlenmeyer synthesis.8,9 The first stereocontrolled synthesis
involved radical chlorination of L-Val, followed by hydrolysis
of the product chlorides to give a mixture of Hyv diastereomers
in very low yields.10 In a second approach selectivity was
achieved via asymmetric radical hydrogen bromide addition.11
Most recently, a catalytic system based on platinum in combina-
tion with copper(II) as oxidant was utilized for C-H bond
functionalization.12 Yields of up to 67% were reported when
L-Val was oxidized in the presence of K2PtCl4 as a catalyst and
CuCl2 as an oxidant. Good regio- and stereoselectivity [3:1 ratio
of (3S,4S) to (3S,4R)] and limited racemization (<5%) was
observed. Isolated L-Hyv lactones were subsequently converted
to Boc-Hyv lactones. However, the Boc-Hyv lactone represented
somewhat of a dead end, as the lactone form is too unreactive
to use for Hyv incorporation into peptide chains. With the need
for the open form of Hyv for peptide synthesis, side chain
hydroxyl protection is also required to prevent lactonization
during activation of the carboxyl group.13,14
To assemble Hyv-containing conopeptides, we have devel-
oped an efficient method for the synthesis of an appropriately
functionalized Fmoc-Hyv building block, Fmoc-D-Hyv(O-
TBDMS). We utilized a variation of the procedure of Sames et
al.12 for the efficient production of D-Hyv from D-Val. Copper-
complexed D-Hyv was prepared and treated with TBDMSOTf
to produce D-Hyv(O-TBDMS). Reaction of D-Hyv(O-TBDMS)
with Fmoc-OSu gave the desired Fmoc-D-Hyv(O-TBDMS).
Fmoc-D-Hyv(O-TBDMS) was characterized by NMR spectros-
copy and mass spectrometry, and used in the synthesis of the
conopeptide gld-V* from Conus gladiator. The isolated synthetic
and natural products had coincidental mass and NMR spectra.
Results and Discussion
Initially, optimized conditions for the catalytic hydroxylation
of Val were developed (Scheme 1). D-Val was treated with
catalytic amounts of K2PtCl4 (10 mol %) in the presence of 10
equiv of CuCl2 in H2O at 160 °C for 16 h. The reaction was
performed in a glass pressure vessel. Dangel et al.12 applied
gaseous H2S for removal of excess CuCl2, which is often used
for the precipitation of the copper as cupric sulfide. A serious
drawback to this method is the H2S toxicity. Moreover, the
reaction with gaseous H2S often requires prolonged heating in
highly acidic conditions to produce manageable precipitates.
Even under those conditions the CuS sometimes stays colloidal
and eludes filtration.15 Thioacetamide had been successfully used
for the decomposition of amino acid copper complexes.15,16 The
alkaline hydrolysis of thioacetamide results in a more tractable
precipitate of CuS that remains readily filterable even upon
subsequent acidification.15 The reaction mixture described above
was treated with thioacetamide under alkaline conditions (pH
8) for 30 min at 110 °C, and then followed by acidic conditions
(pH 2) for an additional 10 min at 110 °C (Scheme 1, route
b1). The insoluble CuS was filtered off upon cooling. We also
explored the use of potassium ferrocyanide for copper precipita-
tion (Scheme 1, route b2). An aqueous solution of potassium
ferrocyanide was added to the reaction mixture and a red-brown
(5) Sharpe, I. A.; Gehrmann, J.; Loughnan, M. L.; Thomas, L.; Adams,
D. A.; Atkins, A.; Palant, E.; Craik, D. J.; Adams, D. J.; Alewood, P. F.;
Lewis, R. J. Nat. Neurosci. 2001, 4, 902-907.
(6) Mar´ı, F.; Fields, G. B. Chim. Oggi 2003, 21 (6), 43-48.
(7) Fu, S.; Hick, L. A.; Sheil, M. M.; Dean, R. T. Free Radical Biol.
Med. 1995, 19, 281-292.
(8) Galantay, E.; Szabo, A.; Fried, J. J. Org. Chem. 1963, 28, 98-102.
(9) Englisch-Peters, S. Tetrahedron 1989, 45, 6127-6134.
(10) Faulstich, H.; Do¨ling, J.; Michl, K.; Wieland, T. Liebigs Ann. Chem.
1973, 560-565.
(11) Easton, C. J.; Merrett, M. C. Tetrahedron 1997, 53, 1151-1156.
(12) Dangel, B. D.; Johnson, J. A.; Sames, D. J. Am. Chem. Soc. 2001,
123, 8149-8150.
(13) Remmer, H. A.; Fields, G. B. In Peptides: Frontiers of Peptide
Science; Proceedings of the Fifteenth American Peptide Symposium, June
14-19, 1997, Nashville, TN; Tam, J. P., Kaumaya, P. T. P., Eds.; Kluwer
Academic Publishers: Dordrecht, The Netherlands, 1999; pp 287-288.
(14) Cudic, M.; Lauer-Fields, J. L.; Fields, G. B. J. Peptide Res. 2005,
65, 272-283.
(15) Taylor, U. F.; Dyckes, D. F.; Cox, J. R., Jr. Int. J. Peptide Protein
Res. 1982, 19, 158-161.
(16) Crivici, A.; Lajoie, G. Can. Synth. Commun. 1993, 23, 49-53.
5582 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis of Fmoc-HyV
SCHEME 2. Synthesis of Fmoc-Hyv(O-TBDMS) with
9-BBN Complexation
precipitate formed. The Cu[Fe(CN)6] precipitate was removed
by centrifugation. In either case (route b1 or b2), evaporation
of the filtrate gave D-Hyv as a mixture of open form isomers
1a and 1b and lactone isomers 1c and 1d (Scheme 1).
A diastereoselective ratio of 3:1 (3S,4S) to (3S,4R) for the
crude reaction material was observed by 1H NMR spectroscopy
in D2O (Supporting Information, Figure 1). The value was based
on the ratio of integration units for the CH3 protons of the
product and unreacted D-Val. The result is in accordance with
that previously published.12 The level of L-Hyv in the product
was not evaluated, as it had been shown previously that
racemization was negligible during the synthesis of L-Hyv from
L-Val.12
Initial attempts to protect the R-amino group of D-Hyv with
the tert-butyl dicarbonate (Boc2O) or benzyl chloroformate (Cbz-
Cl) group (Scheme 1) resulted in low yields. The low yields
could be explained by competing formation of the corresponding
NR-protected Hyv in cyclic (lactone) form and open (linear)
form. It has been shown that the synthesis of N-substituted
homoserines required longer reaction times than N-substitutions
of most amino acids, most likely due to the intramolecular
hydrogen bonding between the amino and the γ-hydroxyl group
of homoserine.17 The purified major isomer of NR-Boc protected
Hyv lactone (2c) and both major and minor isomers of NR-Cbz
protected Hyv lactone (3c and 3d) were isolated and character-
ized by 1H NMR spectroscopy (Supporting Information, Figures
2-4).
An alternative R-amino group protection scheme that included
prior hydroxyl protection was developed to prevent lactone
formation. Previous studies have indicated that 9-borabicy-
clononane (9-BBN) protected amino acids are very tolerant to
a wide range of reaction conditions, and the solubility of the
corresponding borane complexes is significantly improved in
various organic solvents.18-20 The crude reaction mixture of Val
and Hyv was treated with 9-BBN while refluxing in methanol
to give a mixture of soluble borane complexes of Val and Hyv
(Scheme 2) that were successfully separated by column chro-
matography. The hydroxyl group of Hyv was protected by
treatment with tert-butyldimethylsilyl trifluoromethanesulfonate
(TBDMSOTf) in the presence of 2,6-lutidine (Scheme 2). The
major isomer 6a was separated from the minor isomer 6b by
column chromatography. Starting from 4a and 4b, 6a and 6b
were isolated in a combined 88% yield.
Subsequent cleavage of the borane complex proceeded with
more difficulties than expected. Borane complexes of amino
acids are typically cleaved with either aqueous HCl or by
exchange with ethylenediamine in methanol. When 9-BBN was
used for regioselective protection of the R-amino and R-carboxyl
groups of (5R)-δ-hydroxy-L-Lys, complete decomplexation was
reported via treatment with a mixture of chloroform and
methanol over 12 h at room temperature.20 Decomplexation of
the borane complex of Hyv 6a was slow in the presence of
ethylenediamine or a chloroform-methanol mixture and was
observed only after heating the reaction mixture for several hours
at 50 °C. The product 8a was reacted with Fmoc-Cl and the
resulting residue was purified by flash chromatography to
provide Fmoc-D-Hyv(O-TBDMS) 9a in 12% yield starting from
6a. NMR and mass spectra and RP-HPLC analysis confirmed
the purity and composition of Fmoc-D-Hyv(O-TBDMS) (Sup-
porting Information, Figures 5 and 6). The overall yield of 9a
utilizing the 9-BBN complex approach was 2% starting from
D-Val.
Due to the difficulties with 9-BBN complex decomposition,
a copper complex approach was examined (Scheme 3). Copper-
complexed Hyv 5a and 5b was prepared as previously de-
scribed.14 The hydroxyl group of Hyv was protected by
treatment with TBDMSOTf in the presence of DMAP in
pyridine. The desired copper-complexed D-Hyv(O-TBDMS) 7a
and 7b was separated from copper-complexed Val by extraction
with EtOAc-H2O. Disruption of the copper complex was
achieved by using Na+ Chelex 100 resin in a pyridine-H2O
mixture.21 The product 8a and 8b was reacted with Fmoc-OSu
and the resulting residue purified by flash chromatography to
provide Fmoc-D-Hyv(O-TBDMS) 9a and 9b in 56% yield
starting from 8a and 8b. NMR and mass spectra confirmed the
purity and composition of Fmoc-D-Hyv(O-TBDMS) (Supporting
Information, Figures 7 and 8). The overall yield of 9a and 9b
utilizing the copper complex approach was 26% starting from
(17) Ozinskas, A. J.; Rosenthal, G. A. J. Org. Chem. 1986, 51, 5047-
5050.
(18) Dent, W. H., III; Randal Erickson, W.; Fields, S. C.; Parker, M.
H.; Tromiczak, E. G. Org. Lett. 2002, 4, 1249-1251.
(19) Walker, W. H., IV; Rokita, S. E. J. Org. Chem. 2003, 68, 1563-
1566.
(20) Syed, B. M.; Gustafsson, T.; Kihlberg, J. Tetrahedron 2004, 60,
5571-5575.
(21) Barral, I.; Savrda, J. Synthesis 1973, 795-796.
J. Org. Chem, Vol. 72, No. 15, 2007 5583

Cudic et al.
SCHEME 3. Synthesis of Fmoc-Hyv(O-TBDMS) with
Copper Complexation
Information, Figure 13), and MALDI-TOF-MS analysis gave
the desired mass (Supporting Information, Figure 12).
The synthetic gld-V* conopeptide was compared with material
isolated from Conus gladiator. Due to the Hyv chiral center at
the â-carbon, Conus gladiator gld-V* exists as diastereomers,
which are referred to as gld-V* and gld-V*′.3 Prior analysis
indicated that gld-V* corresponds to the Hyv (3S,4S) configu-
ration, whereas gld-V*′ corresponds to the Hyv (3S,4R) con-
figuration.3 Analysis of the synthetic peptide product by using
RP-HPLC conditions designed to separate out gld-V* and gld-
V*′3 showed more than one component of the same mass (data
not shown). NMR spectroscopic analysis found that the most
prominent isolated product corresponded to naturally occurring
gld-V* (Supporting Information, Figure 14). Analysis of the
Fmoc-Hyv(O-TBDMS) derivative used for peptide synthesis
indicated 75% of the (3S,4S) configuration present (see earlier
discussion), and thus it is logical that the most abundant
synthetic peptide diastereomer is the one containing the Hyv
(3S,4S) configuration (gld-V*).
Overall, an efficient 5 step synthesis of an appropriately
functionalized Hyv building block, Fmoc-D-Hyv(O-TBDMS),
has been developed (Scheme 3). Fmoc-D-Hyv(O-TBDMS) was
subsequently utilized for the solid-phase synthesis of a Hyv-
containing conopeptide. The (3S,4S) and (3S,4R) configurations
of Hyv could be separated out following synthesis of either
complexed D-Hyv(O-TBDMS), Fmoc-D-Hyv(O-TBDMS), or
the desired peptide. The methodology presented herein will
greatly facilitate biological studies of Hyv-containing sequences,
such as receptor responses to hydroxylated versus nonhydroxy-
lated conopeptides3 and the relative susceptibility of proteins
to modification by oxidative stress.7
Experimental Section
Synthesis of 4-Hydroxy-D-valine (γ-Hydroxy-D-Val) (1). Syn-
thesis of γ-hydroxy-D-Val was performed as described.12 Briefly,
D-Val (5 mmol, 585 mg) was treated with a catalytic amount of
K2PtCl4 (10 equiv, 10 mol %, 0.5 mmol, 208 mg) in the presence
of CuCl2 × 2H2O (10 equiv, 50 mmol, 8.5 g) and heated to
160 °C for 16 h in a pressure vessel (Chemglass). Copper(II) sulfide
was removed by treatment with thioacetamide or K2PtCl4 (see
below).
(1) Method A: Thioacetamide Removal of Copper(II) Sulfide.
Thioacetamide (1.1 equiv, 55 mmol, 4.25 g) was added to the
reaction mixture followed by NaOH (4 M) until the pH of the
suspension was brought to 8. The suspension was heated at 110
°C for 30 min. The pH of the suspension was brought to 2 with
2 M HCl, followed by heating for 10 min at 110 °C. The reaction
mixture was cooled down and filtered through a bed of Celite. After
filtration, the pH of the solution was brought to 8 and the solution
left in the hood overnight and than evaporated. D-Hyv was obtained
as a mixture of open chain isomers 1a and 1b and lactone isomers
1c and 1d. The product to starting material ratio (×100) as well as
the isomer ratio was determined by 1H NMR spectroscopy
(Supporting Information, Figure 1) as described.12
(2) Method B: Potassium Ferrocyanide Removal of Copper-
(II) Sulfide. The reaction mixture was cooled down to room
temperature. Precipitated CuCl was filtered off and the precipitate
washed well with cold H2O. Potassium ferrocyanide (5 mmol,
2.1 g) was dissolved in H2O (20 mL) and added to the solution.
The red-brown precipitate formed was removed by centrifugation
and then washed two times with H2O. Filtrates were collected and
evaporated to dryness. The residue was dissolved in small amount
of ethanol (10 mL). Nondissolved material (salt) was filtered and
evaporation of filtrate gave D-Hyv as a mixture of open chain
isomers 1a and 1b and lactone isomers 1c and 1d. The product to
D-Val. RP-HPLC analysis (Supporting Information, Figure 9)
of Fmoc-D-Hyv(O-TBDMS) revealed the presence of two peaks
(at 17.7 and 17.9 min) whose areas corresponded well to the
observed diastereoselective ratio of 3:1 (3S,4S) to (3S,4R) for
the D-Hyv mixture of 1a and 1b. By comparison, Fmoc-D-Hyv-
(O-TBDMS) 9a isolated from the 9-BBN complex approach
showed the presence of only one peak by RP-HPLC at 17.7
min (Supporting Information, Figure 6). Fmoc-D-Hyv(O-TB-
DMS) 9a and 9b from the copper complexation approach were
then conveniently separated by preparative RP-HPLC (Sup-
porting Information, Figures 10 and 11) with an overall yield
of 13% starting from D-Val.
The mixture of the major and minor isomers of Fmoc-D-Hyv-
(O-TBDMS) 9a and 9b was used for the solid-phase synthesis
of the D-Hyv containing conopeptide gld-V* (Ala-Hyp-Ala-Asn-
Ser-D-Hyv-Trp-Ser-NH2). The peptide was assembled by au-
tomated Fmoc chemistry. Fmoc-D-Hyv(O-TBDMS) was coupled
by using a 2-fold molar excesses of Fmoc-amino acid and
1-hydroxybenzotriazole (HOBt), a 1.8-fold molar excess of
2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluo-
rophosphate (HBTU), and a 4-fold molar excess of N,N-
diisopropylethylamine. Peptide-resin cleavage and side chain
deprotection proceeded with H2O-thioanisole-TFA (1:1:18)
for 2 h. The peptide was purified by preparative RP-HPLC. The
product was homogeneous by analytical RP-HPLC (Supporting
5584 J. Org. Chem., Vol. 72, No. 15, 2007

Synthesis of Fmoc-HyV
starting material ratio (×100) as well as the isomer ratio was
(2) Method B: Starting from 4-Hydroxy-D-valine Copper
Complex 5a and 5b. A copper-complexed mixture of Val and Hyv
5a and 5b (starting from 5 mmol) was dissolved in pyridine
(20 mL) and TBDMSOTf (10 mmol, 2.2 mL) was introduced in
the presence of DMAP (5 mmol, 604 mg) as catalyst as previously
described for copper-complexed Hyl(Boc).14 After overnight stirring
pyridine was evaporated. The residue was partitioned between
EtOAc and H2O. TLC analysis (n-BuOH-HOAc-H2O, 12:3:5)
of the EtOAc and H2O layers revealed that the desired copper-
complexed D-Hyv(O-TBDMS) 7a and 7b was present in the EtOAc
layer and copper-complexed Val in the H2O layer. EtOAc was
evaporated and the residue was dissolved in a mixture of pyridine
and H2O (1:1, 30 mL). Na+ Chelex 100 resin (10 g) was added to
the stirred solution. After overnight stirring, the resin was filtered
and the colorless solution was evaporated to dryness under reduced
pressure to provide the sodium salt of D-Hyv(O-TBDMS) 8a and
8b (570 mg, 42% yield from 5a and 5b). This material was used
in the next step without further purification.
Synthesis of N-(Fluoren-9-ylmethoxycarbonyl)-4-O-tert-bu-
tyldimethylsilyl-D-valine. D-Hyv(O-TBDMS) 8a and 8b (570 mg,
2.1 mmol) and NaHCO3 (350 mg, 4.2 mmol) were dissolved in
H2O (10 mL), and a solution of Fmoc-OSu (1.06 g, 3.15 mmol) in
acetone (10 mL) was added. Additional acetone (10 mL) was added,
and the reaction proceeded overnight at room temperature. Acetone
was removed under reduced pressure and the remaining H2O
solution was acidified to pH ∼2 with 1 M HCl. The aqueous
suspension was extracted three times with 20 mL of CHCl3. The
CHCl3 layer was washed three times with 20 mL of H2O, dried
over anhydrous sodium sulfate, and concentrated under reduced
pressure. The residue was purified by flash chromatography
(EtOAc-toluene-HOAc, 5:10:1, v/v) to obtain Fmoc-D-Hyv(O-
TBDMS) 9a and 9b (580 mg, 56% yield from 8a and 8b). Fmoc-
D-Hyv(O-TBDMS) 9a and 9b were characterized by 1H NMR
spectroscopy (Supporting Information, Figure 7) and MALDI-TOF-
MS (m/z expected 492.62 [M + Na]+; observed 492.52 [M + Na]+).
A total of 27 mg of Fmoc-D-Hyv(O-TBDMS) 9a and 9b was then
purified by preparative RP-HPLC, yielding 11 mg of 9a and 2 mg
of 9b (Supporting Information, Figures 10 and 11). This represented
an overall yield of 9a and 9b of 13% starting from D-Val.
1
determined by H NMR spectroscopy as described.12
Synthesis of N-tert-Butyloxycarbonyl-4-hydroxy-D-valine (2).
Boc-protected γ-hydroxy-D-Val lactone 2c and 2d was prepared
as described.12 Briefly, the crude reaction mixture of D-Hyv was
dissolved in ethanol (10 mL) and NaOH (2 M) was added until the
pH 10. Boc2O (1.4 equiv, 7 mmol, 1.5 g) was dissolved in THF
(5 mL) and added to the reaction mixture. The reaction was stirred
at room temperature and monitored by TLC (EtOAc-toluene-
HOAc, 5:5:1). Organic solvents were removed by rotary evaporation
before acidification. The reaction was diluted with H2O, cooled to
0 °C, and adjusted to pH 3 with HCl (2 M). The aqueous mixture
was extracted with EtOAc. The organic phase was dried with
anhydrous Na2SO4, filtered, and concentrated to an oil. The product
was a mixture of Boc-Val and Boc-Hyv in lactone and open forms.
This material was purified by flash chromatography (EtOAc-
toluene-HOAc, 5:5:1). The total yield of Boc-Hyv lactone 2c and
2d was 120 mg (10% starting from D-Val). Isolated 2c lactone was
1
characterized by H NMR spectroscopy (Supporting Information,
Figure 2) as described.12
Synthesis of N-Benzyloxycarbonyl-4-hydroxy-D-valine (3).
Cbz-protected γ-hydroxy-D-Val lactone 3c and 3d was prepared
starting from the crude reaction mixture of D-Hyv in H2O. NaOH
(2 M) was added until pH 8 and then Cbz-Cl was added (1.5 equiv,
7.5 mmol, 1080 µL). The reaction was stirred at room temperature
and monitored by TLC (EtOAc-toluene-HOAc, 1:10:1). The
reaction was cooled to 0 °C and adjusted to pH 3 with HCl (2 M).
The aqueous mixture was extracted with EtOAc. The organic phase
was dried with anhydrous Na2SO4, filtered, and concentrated to an
oil. The product was a mixture of Cbz-Val and Cbz-Hyv in lactone
and open forms. This material was purified by flash chromatography
(EtOAc-toluene-HOAc, 1:10:1). The total yield of Cbz-Hyv
lactone 3c and 3d was 120 mg (9% starting from D-Val).
Compounds 3c and 3d were characterized by 1H NMR spectroscopy
(Supporting Information, Figures 3 and 4).
Synthesis of 4-Hydroxy-D-valinatobicyclononylboron 4. The
crude reaction mixture of Val and Hyv (starting from 5 mmol) was
dissolved in methanol (20 mL) and 9-BBN dimer (6 mmol, 1.464
g, 1.2 equiv) was added. The reaction mixture was refluxed for
3 h. Methanol was evaporated and the crude residue was purified
by flash chromatography (EtOAc-toluene, 10:1). Val borane
complex was successfully separated from Hyv borane complex 4a
and 4b. The total yield of Hyv borane complex 4 was 178 mg (14%
starting from 1).
D-Hyv(O-TBDMS) 8a (0.6 mmol), obtained from 9-BBN-
complexed D-Hyv(O-TBDMS) 6a, was reacted with Fmoc-Cl
(1.05 mmol) with use of similar conditions as described above.
The major isomer 9a was isolated in 14% yield starting from 6a
and 2% overall yield starting from D-Val, and characterized by 1H
NMR spectroscopy (Supporting Information, Figure 5).
Synthesis of 4-Hydroxy-D-valine Copper Complex 5. The
copper complex of D-Hyv 5a and 5b was prepared as described.14
Two equivalents of CuCO3 (8-10 mmol) was used and the reaction
was refluxed for 4 h. The reaction mixture was evaporated and
dried well in a desiccator, then was used without further purification.
Synthesis of 4-O-tert-Butyldimethylsilyl-D-valine. (1) Method
A: Starting from 4-Hydroxy-D-valinatobicyclononylboron 4a
and 4b. 9-BBN-complexed Hyv 4a and 4b (180 mg, 0.7 mmol)
were dissolved in a mixture of DCM and THF (2:1, 20 mL), whcih
was cooled to 0 °C, then TBDMSOTf (242 µL, 1 mmol) was
introduced in the presence of 2,6-lutidine (165 µL, 1.38 mmol) as
catalyst.20 The reaction mixture was stirred overnight. The solution
was evaporated and the residue was purified by flash chromatog-
raphy (EtOAc-toluene, 1:1), which allowed for separation of the
major isomer 6a from the minor isomer 6b. The fractions were
collected and after evaporation 230 mg of 6a and 6b combined
was obtained, which corresponded to 88% yield starting from 4a
and 4b. The major isomer (6a) was dissolved in a mixture of
methanol and chloroform (1:5, 10 mL) and the solution stirred
overnight at room temperature. There was no noticeable decom-
position of the complex; therefore, the reaction mixture was stirred
for an additional 16 h at 50 °C. The solution containing decom-
plexed D-Hyv(O-TBDMS) 8a was evaporated to dryness under
reduced pressure and the residue was used in the next step without
further purification.
Peptide Synthesis. Peptide-resin assembly was performed on
an automated peptide synthesizer by using Rink amide MBHA resin
with an initial load of 0.72 mmol/g. Standard Fmoc chemistry was
used throughout with a 4-fold molar excess of the acylating amino
acids, and HBTU and HOBt as coupling reagents.22 Fmoc-D-Hyv-
(O-TBDMS) 9a and 9b were coupled manually in 2-fold molar
excess to reduce consumption of this amino acid. The peptide was
cleaved from the resin with H2O-thioanisole-TFA (1:1:18) for
2 h. The peptide (Ala-Hyp-Ala-Asn-Ser-D-Hyv-Trp-Ser-NH2) was
purified by preparative RP-HPLC. The product was homogeneous
by analytical RP-HPLC and MALDI-TOF-MS analysis gave the
desired mass: m/z expected 862.89 [M + H]+; observed 863.49
[M + H]+ and 885.48 [M + Na]+. Diastereomeric peptides were
separated by using C18 RP-HPLC.3 Initially, peptides were separated
with an elution gradient of 0-100% B over 100 min where A was
0.1% TFA in H2O and B was 0.1% TFA in CH3CN-H2O (3:2).
Isolated fractions were then further purified via isocratic elution at
20.3% B. One-dimensional 1H NMR spectra of the most prominent
diastereromeric peptide and gld-V* isolated from Conus gladiator
were recorded at 25 °C with use of a 1.7 mm tube in a 3 mm gHCN
probe. The NMR assignments of the γ-hydroxy-D-Val (HN: d 7.99,
(22) Fields, C. G.; Lloyd, D. H.; Macdonald, R. L.; Otteson, K. M.;
Noble, R. L. Peptide Res. 1991, 4, 95-101.
J. Org. Chem, Vol. 72, No. 15, 2007 5585

Cudic et al.
8 Hz; RH: 4.45, m; γCH2: m 3.15; âCH: m 1.89, 6.9 Hz; γCH3:
d 0.52, 7.1 Hz) were in accordance with the reported values of the
synthetic amino acid.11
Supporting Information Available: General experimental
methods; one-dimensional H NMR spectra of D-Hyv 1, Boc-D-
1
Hyv lactone 2c, Cbz-D-Hyv lactone 3c and 3d, Fmoc-D-Hyv(O-
TBDMS) 9a, Fmoc-D-Hyv(O-TBDMS) 9a and 9b, and isolated and
synthesized gld-V*; RP-HPLC analysis of Fmoc-D-Hyv(O-TBDMS)
9a and 9b, Fmoc-D-Hyv(O-TBDMS) 9a, Fmoc-D-Hyv(O-TBDMS)
9b, and synthetic gld-V*; and MALDI-TOF mass spectra of Fmoc-
D-Hyv(O-TBDMS) 9a and 9b and synthetic gld-V*. This material
is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. We thank Sanaz Dovell for the RP-HPLC
isolation of the native and synthetic gld-V*. This work was
supported by the Florida Sea Grant College Program (R/LR-
MB-18), the National Institutes of Health (GM 066004 to F.M.,
CA 77402 to G.B.F.), and the FAU Center of Excellence in
Biomedical and Marine Biotechnology. An acknowledgment is
also made to the National Science Foundation Division of
Undergraduate Education (0311369) for the 400 MHz NMR
instrument used in this study.
JO070436Y
5586 J. Org. Chem., Vol. 72, No. 15, 2007