A synthetic pathway to Cys-Xxx-Cys (N2S2) analogue ligands: An improved synthesis of HSCH2CH2C(O)NHCH 2C(O)NHCH2CH2SH

Article abstract of DOI:10.1080/10426500600614824

Herein, two improved synthetic pathways to biologically relevant Cys-Xxx-Cys analogue ligands used in conjunction with metals as metalloenzyme models (Ni for carbon monoxide dehydrogenase/acetyl-CoA synthase A-cluster; Co and Fe for nitrile hydratase) are reported. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426500600614824

Phosphorus, Sulfur, and Silicon, 181:2263–2272, 2006
Copyright © Taylor & Francis Group, LLC
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426500600614824
A Synthetic Pathway to Cys-Xxx-Cys (N2S2) Analogue
Ligands: An Improved Synthesis of
HSCH2CH2C(O)NHCH2C(O)NHCH2CH2SH
Jennifer K. Angelosante
Breia J. Lewis
Lisa E. Cooper
Rebecca A. Swanson
Christopher J. A. Daley
Department of Chemistry, Western Washington University,
Bellingham, Washington
Herein, two improved synthetic pathways to biologically relevant Cys-Xxx-Cys ana-
logue ligands used in conjunction with metals as metalloenzyme models (Ni for
carbon monoxide dehydrogenase/acetyl-CoA synthase A-cluster; Co and Fe for ni-
trile hydratase) are reported.
Keywords Carboxamido ligand; metal thiolate; peptide synthesis; synthetic analogue
INTRODUCTION
Over the past decade, examples of backbone amide nitrogen bonds
to transition metal centers have been observed in metalloenzymes.
While this is not a common binding mode, there exists three known
cases where such binding occurs: (1) the A-Cluster in carbon monoxide
dehydrogenase/acetyl-CoA synthase (CODH/ACS),1 (2) the P-Cluster
in nitrogenase,2 and (3) the catalytic core of Fe(III) and Co(III) ni-
trile hydratase (NHase).3 To gain a better understanding of these
Received November 20, 2005; accepted January 4, 2006.
Funding for this research was provided by the Research Corporation Cottrell College
Grant 6600, American Philosophical Society; Franklin Research grant; and the Bureau
of Faculty Research at Western Washington University. The authors would like to thank
the Dreyfus Foundation: Faculty Startup Award (glovebox and solvent purification sys-
tem) and the National Science Foundation: MRI grant NSF-0216604 (500 MHz NMR)
for equipment funding. Ms. Angelosante would like to thank the Dreyfus Foundation for
funding through the Jean Dreyfus Boissevain Undergraduate Scholarship.
Address correspondence to Christopher J. A. Daley, Western Washington Univer-
sity, Department of Chemistry, 516 High Street, Bellingham, WA 98225. E-mail: daley@
chem.wwu.edu
2263

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J. K. Angelosante et al.
FIGURE 1 Depictions of the active sites of CODH/ACS (left) and NHase (right)
that contain the Cys-Xxx-Cys peptide fragment coordinated to the metal center
through the cysteine sulfurs and two backbone amide nitrogens.
biological systems, groups have used the synthetic analogue approach
to synthesize active models of the catalytic centers of these metal-
loenzymes. In the case of CODH/ACS and the Fe- and Co-NHase, the
catalytic center is bound by a Cys-Xxx-Cys fragment in a tetraden-
tate plane from the two cysteine thiolates and the backbone amide
nitrogen of the central amino acid (Xxx: Gly for CODH/ACS; Ser for
NHase) and the adjacent cysteine (Figure 1). As such, synthetic routes
to Cys-Xxx-Cys analogues are important. Herein we report an im-
proved synthetic approach to obtain the generic Cys-Xxx-Cys analogue
HSCH2CH2C(O)NHCH2C(O)NHCH2CH2SH (1) that can also be used
to obtain other modified analogues of the Cys-Xxx-Cys form.
RESULTS AND DISCUSSION
Rao and colleagues recently reported4 the synthesis of 1 and its use
as a synthetic analogue ligand for the nickel binding domain of the A-
Cluster of CODH/ADS. The synthetic scheme was based on an earlier
report for a similar ligand that entailed 7 steps and an overall 22%
yield. The resultant product was described as an ether or water solvate
based on elemental analysis. Our group’s efforts to develop synthetic
analogues resulted in the design of a number of Cys-Xxx-Cys type ana-
logues that included the ligand reported by Rao and colleagues. Here we
report an improved synthesis of 1 that reduces the number of synthetic
steps by one (from 7 to 6) and increases the overall yield depending
on the protecting groups utilized (S-benzyl = 33%; S-tert-butyl = 57%).
Scheme 1 depicts the synthetic pathway used starting from either ben-
zyl or tert-butyl protected 3-mercaptopropionic acid (2). The peptide cou-
ple of 2-R (R = Bn or tBu) with 3 utilized HBTU as a coupling reagent
and DMF as a solvent. The products 5-R (R = Bn or tBu) were obtained
in high yields, and their subsequent benzyl ester cleavage produced
6-R (R = Bn or tBu). When using the tert-butyl protected pathway, the

A Synthetic Pathway to Cys-Xxx-Cys (N S ) Analogue Ligands
2265
2
2
t
SCHEME 1 (a) BrBn, NaOH, EtOH. (b) H2SO4, BuOH, 0◦C to 25◦C, 3 h.
(c) HBTU, DIEPA, DMF. (d) AlCl3, EtSH. (e) K2CO3, MeOH:H2O, reflux, 16 h.
(f) BrBn, K2CO3, EtOH. (g) HCl, tBuOH, reflux, 16 h. (h) HBTU, DIPEA, DMF.
(i) NH3, Na, THF, −78◦C. (j) i. Hg(OAc)2, TFA; ii. H2S, MeCN.
use of K2CO3 to deprotect the carboxylic acid resulted in a high yield
of product. However, under the same conditions, the benzyl ester was
not cleaved from 5-Bn owing to its insolubility in the reaction media.
In order to dissolve 5-Bn, a ratio of greater than 1:1 methanol:water
was required. As reported earlier,5 when the methanol:water ratio is
greater than 70:30, reaction does not proceed cleanly. The reaction of
5-Bn with AlCl3 in ethanethiol was successful in cleaving the benzyl
ester group providing an overall high yield. Once 6-Bn and 6-tBu were
obtained, they were coupled under similar conditions used to make 5-R.
6-R was coupled with its corresponding protected thioamine 4-R, where
the same protecting groups were used (e.g., 6-Bn + 4-Bn), resulting in
the di-S-benzyl or di-S-tert-butyl protected ligands 7 (7-Bn-Bn and 7-
tBu-tBu, respectively). The desired analogue 1 was obtained by either
the dissolving metal reduction (from 7-Bn-Bn) or the mercuric acetate
in trifluoroacetic acid (from 7-tBu-tBu) protocols. In each case, 1 was
obtained as an off-white solid in good to excellent yields. This synthetic
pathway is currently being used in our laboratory to synthesize a vari-
ety of N2S2 ligands by varying the amino acid (3) core.
EXPERIMENTAL
General
All materials were purchased from suppliers and used without further
purification unless stated otherwise. All reactions were performed us-
ing standard Schlenk techniques unless stated otherwise. 1H NMR data

2266
J. K. Angelosante et al.
were obtained on a Varian Mercury 300 MHz or INOVA 500 MHz NMR
spectrometer. Chemical shifts for protons are referenced to tetramethyl-
silane (TMS) at 0.00 ppm. S-benzyl-2-aminoethanethiol (4-Bn) was syn-
thesized using a literature procedure in 90% yield6 and was converted
to the HCl salt for prolonged storage.
The Synthesis of 2-Bn
This reaction was performed in air. A 1 M NaOH solution (130 mL) was
added to a 500-mL round-bottom flask along with ethanol (130 mL).
3-mercaptopropionic acid (2; 4.93 mL, 56.5 mmol) was subsequently
added dropwise via a syringe to the reaction flask. The solution was
cooled to 0◦C and stirred for 5 min before benzyl bromide (7.06 mL,
59.4 mmol) was added dropwise via a syringe. After 10 min, the solu-
tion was allowed to warm to r.t. and stirred for a further 3 h. A 1 M HCl
solution was added to the reaction flask to adjust the pH to 1, and the
resulting solution was transferred to a 500-mL separatory funnel, and
extracted with CH2Cl2 (5 × 30 mL). A 1 M NaOH solution was added
to the combined organic phase to adjust the pH to 12. The solution was
extracted with CH2Cl2 (2 × 30 mL). The aqueous layer was acidified
to pH = 1 with 1 M HCl and extracted with CH2Cl2 (4 × 20 mL). The
CH2Cl2 layer was dried over magnesium sulfate, filtered, and concen-
trated yielding 2-Bn as a white solid (9.43 g, 48.1 mmol, 85.0% yield).
1H NMR (CDCl3, 500 MHz): δ2.61 (t, 2H, CH CH2SCH2Ar), 2.69 (t,
2
2H, CH2CH SCH2Ar), 3.75 (s, 2H, CH2CH2SCH Ar), 7.24–7.34 (m, 5H,
2
2
ArH). Anal. calcd. for C10H12O2S: C, 61.20; H, 6.16. Found: C, 61.46; H,
6.17.
The Synthesis of 5-Bn
3-Benzylsulfanyl-propionic acid (2-Bn; 4.00 g, 20.4 mmol) was added to
a 100-mL round-bottom flask equipped with a stirbar, followed by the
addition of NH3-Gly-OBnz· tosylate (3; 6.88 g, 20.4 mmol). Dry DMF
(25 mL) was added to the flask, forming a light yellow solution, fol-
lowed by the addition of diisopropylethylamine (10.65 mL, 61.13 mmol).
HBTU (8.50 g, 22.4 mmol) was added to a separate 50-mL round-bottom
flask equipped with a stirbar along with dry DMF (25 mL). The HBTU
solution was added via cannula to the reaction flask. The solution was
stirred for 16 h at r.t. under a nitrogen atmosphere. The solution was
transferred to a 500-mL separatory funnel, and the organic phase was
washed with 1 M HCl (5 × 20 mL), saturated sodium bicarbonate solu-
tion (5 × 20 mL), and deionized water (5 × 20 mL). The organic phase
was dried over magnesium sulfate, filtered, and concentrated, affording

A Synthetic Pathway to Cys-Xxx-Cys (N S ) Analogue Ligands
2267
2
2
5-Bn as a peach solid (6.81 g, 19.8 mmol, 97.0% yield). 1H NMR (CDCl3,
500 MHz): δ2.42 (t, 2H, CH CH2SCH2Ar), 2.72 (t, 2H, CH2CH SCH2Ar),
2
2
3.72 (s, 2H, CH2CH2SCH Ar), 4.06 (d, 2H, CH2CONHCH CO), 5.18 (s,
2
2
2H, CH2CONHCH2COOCH Ar), 6.19 (br t, 1H, CH2CONHCH2), 7.22–
2
7.84 (m, 10H, ArH). Anal. calcd. for C19H21NO3S: C, 66.45; H, 6.16; N,
4.08. Found: C, 66.47; H, 6.42; N, 4.41.
The Synthesis of 6-Bn
AlCl3 (2.575 g, 19.24 mmol) was added to a 300-mL round-bottom flask
under nitrogen atmosphere and dissolved in ethanethiol (17 mL) at 0◦C.
5-Bn (2.438 g, 7.100 mmol) was added to the solution, and the solution
immediately turned bright yellow. The solution was stirred at 0◦C for
30 min and for a further 2.5 h at r.t. Deionized water (40 mL) and 4 M
HCl (40 mL) were added to the solution slowly and stirred until the
AlCl3 stopped reacting and the solution had a pH = 1. Note: After the
addition of water, a white precipitate formed, added acid dissolved the
precipitate. The solution was transferred to a 250-mL separatory fun-
nel, and the aqueous layer was extracted with CH2Cl2 (6 × 20 mL).
The combined organic phases were subsequently washed with brine
(3 × 20 mL) then dried over magnesium sulfate. The solution was fil-
tered and concentrated yielding a residue that was treated with hex-
anes (3 × 20 mL) and filtered. The product 6-Bn was collected as a white
solid (1.529 g, 6.035 mmol, 85.00% yield). 1H NMR (500 MHz, CDCl3):
δ2.45 (t, 2H, CH CH2SCH2Ar), 2.74 (t, 2H, CH2CH SCH2Ar), 3.74
2
2
(s, 2H, CH2CH2SCH Ar), 4.03 (d, 2H, NHCH COOH), 6.21 (br t, 1H,
2
2
NHCH2COOH), 7.20–7.35 (m, 5H, ArH). Anal. calcd. for C12H15NO3S:
C, 56.90; H, 5.97; N, 5.53. Found: C, 56.98; H, 6.12; N, 5.65.
The Synthesis of 7-Bn-Bn
6-Bn (0.3010 g, 1.188 mmol) was added to a 50-mL round-bottom flask
equipped with a stirbar, followed by the addition of the 4-Bn · HCl
(0.1759 g, 0.8634 mmol) under a nitrogen atmosphere. Dry DMF (25 mL)
was added followed by the addition of dry diisopropylethylamine
(450 µL, 2.58 mmol) via a syringe. HBTU (0.3620 g, 0.9545 mmol)
was dissolved in DMF (10 mL) in a separate 25-mL round-bottom flask
equipped with a stirbar under a nitrogen atmosphere. The HBTU so-
lution was added to the reaction flask via cannula, and the solution
was stirred for 15 h at r.t. The solution was transferred to a 250-mL
separatory funnel, and the organic phase was washed with a 1 M
HCl (5 × 10 mL), saturated sodium bicarbonate solution (5 × 10 mL),

2268
J. K. Angelosante et al.
and deionized water (5 × 10 mL). The organic phase was dried over
magnesium sulfate for 16 h, filtered, and concentrated, affording
7-Bn-Bn as a cream colored solid (0.2334 g, 0.5698 mmol, 66.00%).
1H NMR (500 MHz, CDCl3): δ2.40 (t, 2H, COCH CH2S), 2.57 (t, 2H,
2
NHCH2CH S), 2.77 (t, 2H, COCH2CH S), 3.40 (q, 2H, NHCH CH2S),
2
2
2
3.71 (s, 2H, SCH Ar), 3.73 (s, 2H, SCH Ar), 3.86 (d, 2H, CONHCH CO),
2
2
2
6.05 (br t, 1H, NHCH2CO), 6.33 (br t, 1H, NHCH2CH2S), 7.24–7.36 (m,
10H, ArH). Anal. calcd. for C21H26N2O2S2: C, 62.65; H, 6.51; N, 6.96.
Found: C, 62.92; H, 6.85; N, 6.75.
The Synthesis of the Cys-Xxx-Cys Analogue Ligand (1) From
Benzyl Protected Thiols
Liquid NH3 (50 mL) was condensed into a 250-mL flask cooled in an
isopropyl alcohol/dry ice bath. Sodium metal (0.0400 g, 1.74 mmol) was
added to the ammonia in small portions until the solution remained
deep blue. The protected ligand 7-Bn-Bn (0.1812 g, 0.4501 mmol) was
dissolved in THF (10 mL) in a 50-mL round-bottom flask under a nitro-
gen atmosphere. The THF solution was slowly delivered by cannula
into the ammonia solution. Excess sodium (0.080 g, 3.5 mmol) was
used in order to maintain the blue color for 45 min. After an hour of
stirring, the solution was quenched with ammonium chloride (2.00 g,
37.4 mmol). This mixture was stirred for 5 min, then the dry ice bath
was removed, and the ammonia evaporated over 1–2 h with the aid
of a flow of nitrogen gas. The residue was reverse extracted with THF
(4 × 15 mL), and the THF filtrate concentrated under vacuum to yield 1
as a white solid (0.0781 g, 0.352 mmol, 78.1% yield). 1H NMR (500 MHz,
CDCl3): δ1.40 (t, 1H, NHCH2CH2SH), 1.66 (t, 1H, COCH2CH2SH),
2.58 (t, 2H, COCH CH2SH), 2.68 (td, 2H, NHCH2CH SH), 2.85
2
2
(td, 2H, COCH2CH SH), 3.47 (q, 2H, NHCH CH2SH), 3.98 (d, 2H,
2
2
CONHCH CO), 6.42 (br t, 1H, CONHCH2CO), 6.61 (br t, 1H,
2
NHCH2CH2SH). Anal. calcd. for C7H14N2O2S2: C, 37.82; H, 6.35; N,
12.60. Found: C, 37.68; H, 6.80; N, 12.25.
The Synthesis of 2-tBu
This reaction was performed in air. Deionized water (100 mL) was
added to a 500-mL round-bottom flask, followed by the dropwise ad-
dition of concentrated sulfuric acid (160 mL). The solution was stirred
at 0◦C in an ice/water bath for 5 min. Tert-Butanol (80.0 mL, 0.837 mol)
was added dropwise, followed by the addition of 3-mercaptopropionic
acid (8.20 mL, 0.941 mmol). This solution was stirred for 10 min
at 0◦C then warmed to r.t. and stirred for an additional 3 h. The

A Synthetic Pathway to Cys-Xxx-Cys (N S ) Analogue Ligands
2269
2
2
solution was quenched with ice (300 g) and transferred to a 1-L separa-
tory funnel, and the aqueous phase was washed with Et2O (4 × 50 mL).
The aqueous phase was then made basic with 2 M NaOH so the pH = 12.
The aqueous phase was washed with CH2Cl2 (3 × 50 mL) then re-
acidified to pH = 2 and extracted with CH2Cl2 (3 × 25 mL). The lat-
ter combined organic phases were washed with brine (2 × 50 mL) and
deionized water (1 × 20 mL), dried over magnesium sulfate, filtered,
and concentrated, affording a yellow liquid. On standing in air, the
liquid crystallized into colorless crystals of 2-tBu (14.8 g, 0.912 mol,
96.9% yield). In subsequent syntheses, seed crystals were added to
1
the oil to promote crystallization. H NMR (CDCl3, 500 MHz): δ1.34
(s, 9H, CH2CH2SC(CH ) ), 2.64 (t, 2H, CH CH2SC(CH3)3), 2.80 (t, 2H,
3 3
2
CH2CH SC(CH3)3). Anal. calcd. for C7H14O2S: C, 62.83; H, 6.71. Found:
2
C, 62.83; H, 6.76.
t
r
The Synthesis of 4- Bu HCl
This reaction was performed in air. β-mercaptoethylamine·
hydrochloride (15.20 g, 0.1337 mol) and tert-butanol (16.6 mL,
0.174 mol) were added to a 250-mL round-bottom flask. Two M HCl
(50 mL) was added, which dissolved the salt and made the solution
clear and colorless. The reaction mixture was refluxed overnight
at 100◦C. The reaction mixture was cooled to r.t. and concentrated
revealing a white solid. The product was filtered, washed, and dried
with acetone to remove any water. 4-tBu · HCl was obtained as a white
1
solid (19.88 g, 0.1171 mol, 87.58% yield). H NMR (500 MHz, D2O):
δ1.19 (s, 9H, CH2CH2SC(CH ) ), 2.72 (t, 2H, CH2CH SC(CH3)3), 3.04
3 3
2
(br t, 2H, CH CH2SC(CH3)3). Anal. calcd. for C6H16ClNS: C, 42.46; H,
2
9.50. Found: C, 42.65; H, 9.86.
The Synthesis of 5-tBu
Protected thiol 2-tBu (4.01 g, 24.7 mmol) was added to a 100-mL
round-bottom flask equipped with a stirbar, followed by the addi-
tion of NH3-Gly-OBn· tosylate (3; 6.93 g, 20.5 mmol) under a nitro-
gen atmosphere. Dry DMF (25 mL) was added to the flask contain-
ing the protected thiol and protected glycine via a syringe, thereby
forming a light yellow solution followed by the addition of dry diiso-
propylethylamine (10.7 mL, 61.6 mmol) via a syringe. HBTU (8.56 g,
22.6 mmol) was added to a separate 50-mL round-bottom flask, and
dissolved in DMF (25 mL) under a nitrogen atmosphere. The HBTU
solution was subsequently added via cannula to the reaction flask,
and the reaction mixture was stirred overnight at r.t. The solution

2270
J. K. Angelosante et al.
was transferred to a 500-mL separatory funnel, and the organic phase
was washed with a 1 M HCl solution (5 × 20 mL), saturated sodium
bicarbonate solution (5 × 20 mL), and de-ionized water (2 × 10 mL).
The organic layer was dried overnight with magnesium sulfate, fil-
tered, and concentrated, affording viscous oil (5.76 g, 18.6 mmol, 90.7%).
1H NMR (CDCl3, 500 MHz): δ1.33 (s, 9H, CH2CH2SC(CH ) ), 2.50 (t,
3 3
2H, CH CH2SC(CH3)3), 2.83 (t, 2H, CH2CH SC(CH3)3), 4.11 (d, 2H,
2
2
CONHCH CO), 5.20 (s, 2H, CO2CH Ar), 6.18 (br t, 1H, CONHCH2CO),
2
2
7.33–7.40 (m, 5H, ArH). Anal. calcd. for C16H23NO3S: C, 62.11; H, 7.49;
N, 4.53. Found: C, 61.91; H, 7.75; N, 4.58.
The Synthesis of 6-tBu
This reaction was performed in air. Protected ester 5-tBu (4.67 g,
15.1 mmol) and potassium carbonate (4.18 g, 30.3 mmol) were added
to a 300-mL round-bottom flask with methanol (75 mL) and deion-
ized water (75 mL). The solution was stirred for 10 min, and then
additional potassium carbonate (2.79 g, 20.2 mmol) was added. Ad-
ditional methanol (30 mL) was added to dissolve all solids. Note: the
reaction did not proceed when less than 30% (v/v) water in ethanol
was used. The solution was refluxed for 16 h and then transferred to
a separatory funnel. The reaction mixture was extracted with CH2Cl2
(3 × 25 mL). The aqueous phase was made acidic (pH = 1) by the addi-
tion of 4 M HCl (30 mL), and then the solvent was removed under vac-
uum to yield an oil-solid residue. The residue was extracted with Et2O
(3 × 50 mL), and the Et2O was removed under vacuum to yield 6-tBu
as a white solid (2.98 g, 13.6 mmol, 90.1%). 1H NMR (500 MHz, CDCl3):
δ 1.33 (s, 9H, CH2CH2SC(CH ) ), 2.50 (t, 2H, CH CH2SC(CH3)3), 2.80
3 3
2
(t, 2H, CH2CH SC(CH3)3), 4.10 (d, 2H, CONHCH CO), 6.40 (br t, 1H,
2
2
CONHCH2CO). Anal. calcd for C9H17NO3S: C, 49.29; H, 7.81; N, 6.39.
Found: C, 48.95; H, 8.04; N, 6.19.
The Synthesis of 7-tBu-tBu
Deprotected ester 6-tBu (11.1 g, 50.6 mmol) was added to a 500-mL
round-bottom flask equipped with a stirbar, followed by the addition of
the 4-tBu·HCl (8.59 g, 50.6 mmol) under nitrogen atmosphere. DMF
(50 mL) was added to the flask followed by the addition of diisopropy-
lethylamine (780 µL, 4.49 mmol) via a syringe yielding a yellow col-
ored solution. HBTU (21.1 g, 55.7 mmol) was added to a separate 250-
mL round-bottom flask and dissolved in DMF (80 mL) under-nitrogen
atmosphere. The HBTU solution was added via cannula to the re-
action flask, and the pale gold-colored solution was allowed to react
overnight at r.t. The solution was transferred to a 500-mL separatory

A Synthetic Pathway to Cys-Xxx-Cys (N S ) Analogue Ligands
2271
2
2
funnel, and the organic phase was washed with a 1 M HCl solution (5 ×
50 mL), and saturated sodium bicarbonate solution (5 × 50 mL), brine
(3 × 50 mL), and deionized water (2 × 50 mL). The organic phase was
dried overnight with magnesium sulfate, filtered, and concentrated, af-
1
fording 7-tBu-tBu as a viscous oil (16.08 g, 48.07 mmol, 95.0%). H
NMR (CDCl3, 500 MHz): δ1.33 (s, 9H, NHCH2CH2SC(CH ) ), 1.34 (s,
3 3
9H, COCH2CH2SC(CH ) ), 2.51 (t, 2H, COCH CH2SC(CH3)3), 2.69 (t,
3 3
2
2H, NHCH2CH SC(CH3)3), 2.86 (t, 2H, COCH2CH SC(CH3)3), 3.46 (q,
2
2
2H, NHCH CH2SC(CH3)3), 3.95 (d, 2H, CONHCH CO), 6.30 (br t, 1H,
2
2
CONHCH2CO), 6.42 (br t, 1H, NHCH2CH2SC(CH3)3). Anal. calcd. for
C15H30N2O2S2: C, 53.85; H, 9.04; N, 8.37. Found: C, 53.45; H, 9.46; N,
8.79.
The Synthesis of the Cys-Xxx-Cys Analogue Ligand (1) From
tert-Butyl Protected Thiols
The protected ligand 7-tBu-tBu (4.00 g, 12.0 mmol) was added to a
100-mL round-bottom side-arm flask under nitrogen. Trifluoroacetic
acid (25 mL) and anisole (0.1 mL) were added, and the flask was cooled
to 0◦C. Mercuric acetate (8.01 g, 25.1 mmol) was added as a solid to
the solution, which turned dark yellow on addition. The mixture was
allowed to stir at 0◦C for 1 h, after which time, the solution was bur-
gundy colored. The solvent was removed under vacuum, and the residue
was redissolved in acetonitrile (25 mL). The solution was then bubbled
with hydrogen sulfide gas for 30 min, ensuring all mercury had reacted,
yielding a black precipitate. The solution was filtered through a Celite
column (diam. × height = 1.5ꢀꢀ × 2ꢀꢀ; well packed) and washed with ace-
tonitrile (50 mL), followed by THF (300 mL). The clear, yellow filtrate
was concentrated, yielding an off-white paste. The residue was washed
and filtered with Et2O (4 × 20 mL) to yield 8 as a white solid (2.32 g,
10.4 mmol, 86.7%). Analysis indicated that the product 1 obtained was
identical to that described for the synthesis of 1 from 7-Bn-Bn.
CONCLUSIONS
The synthesis of analogue biologically relevant peptide fragments is
of great importance owing to the increased understanding of native
enzymes using the synthetic analogue approach. As such, new and/or
better methods to synthesize key binding fragments are of continued
interest to the synthetic community. The reported new synthetic path-
way incorporates two different thiol-protecting groups and could thus
be suitably used to design modified Cys-Xxx-Cys ligands that may be
sensitive to the cleavage method of one of the protecting groups. Overall,

2272
J. K. Angelosante et al.
this strategy will allow for the development of many new analogue lig-
ands in good to excellent yields.
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