Preparation and applications of xanthenylamide (XAL) handles for solid-phase synthesis of C-terminal peptide amides under particularly mild conditions

Article abstract of DOI:10.1021/jo960312d

[[9-[(9-Fluorenylmethyloxycarbonyl)amino]xanthen-2(or 3)-yl]oxy]alkanoic acid (XAL) handles have been prepared by efficient four-step routes from 2- or 3-hydroxyxanthone and coupled onto a range of amino-functionalized supports. The resultant XAL supports are the starting points for solid-phase peptide synthesis by Fmoc chemistry. Upon completion of chain assembly, C-terminal peptide amides are released in excellent yields and purities by use of low concentrations [1-5% (v/v)] of trifluoroacetic acid (TFA) in dichloromethane, often without a need for added carbocation scavengers. These cleavage conditions allow retention of all or a significant portion of tert-butyl type and related side-chain protecting groups, which subsequently may be removed fully in a solution process carried out at higher acid concentration. XAL supports are particularly useful for the synthesis of acid-sensitive peptides, including tryptophan-containing sequences that are known to be susceptible to yield- and/or purity-reducing alkylation side reactions. The effectiveness of this chemistry was shown with the syntheses of prothrombin (1-9), acyl carrier protein (65-74), Tabanus atratus adipokinetic hormone, fragments of the protein RHK 1, CCK-8 sulfate, and oxytocin. Furthermore, the application of XAL supports for the preparation of fully protected peptide amides has been demonstrated.

Full text of DOI:10.1021/jo960312d

6326
J . Org. Chem. 1996, 61, 6326-6339
P r ep a r a tion a n d Ap p lica tion s of Xa n th en yla m id e (XAL) Ha n d les
for Solid -P h a se Syn th esis of C-Ter m in a l P ep tid e Am id es u n d er
P a r ticu la r ly Mild Con d ition s1-3
Yongxin Han,4a Susan L. Bontems,4a,5a Peter Hegyes,4a,5b Mark C. Munson,4a,5c
Charles A. Minor,4b,5d Steven A. Kates,4b Fernando Albericio,*, 4a-c and George Barany*, 4a
Department of Chemistry, University of Minnesota, 207 Pleasant Street S.E.,
Minneapolis, Minnesota 55455, PerSeptive Biosystems Biosearch Products, 500 Old Connecticut Path,
Framingham, Massachusetts 01701, and Department of Organic Chemistry, University of Barcelona,
08028-Barcelona, Spain
Received February 14, 1996X
[[9-[(9-Fluorenylmethyloxycarbonyl)amino]xanthen-2(or 3)-yl]oxy]alkanoic acid (XAL) handles have
been prepared by efficient four-step routes from 2- or 3-hydroxyxanthone and coupled onto a range
of amino-functionalized supports. The resultant XAL supports are the starting points for solid-
phase peptide synthesis by Fmoc chemistry. Upon completion of chain assembly, C-terminal peptide
amides are released in excellent yields and purities by use of low concentrations [1-5% (v/v)] of
trifluoroacetic acid (TFA) in dichloromethane, often without a need for added carbocation scavengers.
These cleavage conditions allow retention of all or a significant portion of tert-butyl type and related
side-chain protecting groups, which subsequently may be removed fully in a solution process carried
out at higher acid concentration. XAL supports are particularly useful for the synthesis of acid-
sensitive peptides, including tryptophan-containing sequences that are known to be susceptible to
yield- and/or purity-reducing alkylation side reactions. The effectiveness of this chemistry was
shown with the syntheses of prothrombin (1-9), acyl carrier protein (65-74), Tabanus atratus
adipokinetic hormone, fragments of the protein RHK 1, CCK-8 sulfate, and oxytocin. Furthermore,
the application of XAL supports for the preparation of fully protected peptide amides has been
demonstrated.
Work from our laboratories6,7 and others8 has provided
several methods for the stepwise solid-phase synthesis
of C-terminal peptide amides in conjunction with protec-
tion schemes using the base-labile NR-9-fluorenylmeth-
yloxycarbonyl (Fmoc) function.9 The best approaches use
appropriate handles,10 which are in effect N-protected
(aromatic) amino acids that can be coupled onto amino-
functionalized supports and later serve as a starting point
X Abstract published in Advance ACS Abstracts, J uly 15, 1996.
(1) Abbreviations: AA, amino acid residue (free or protected,
depending on context); BOP, (benzotriazolyloxy)tris(dimethylamino)-
phosphonium hexafluorophosphate; CCK-8, cholecystokinin octapep-
tide (residues 26-33); DME, 1,2-dimethoxyethane; DIPCDI, N,N′-
diisopropylcarbodiimide; DIEA, N,N-diisopropylethylamine; DMF, N,N-
dimethylformamide; EtOAc, ethyl acetate; FABMS, fast atom
bombardment mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbo-
nyl; Fmoc-NH2, 9-fluorenylmethyl carbamate; HOAc, acetic acid; HOAt,
7-aza-1-hydroxybenzotriazole; HOBt, 1-hydroxybenzotriazole; HATU,
N-[(dimethylamino)(1H-1,2,3-triazolo[4,5-b]pyridin-1-yl)methylene]-N-
methylmethanaminium hexafluorophosphate N-oxide; HPLC, high-
performance liquid chromatography; MBHA, p-methylbenzhydry-
lamine (resin); NMM, N-methylmorpholine; PAL, 5-[[(4-Fmoc-
amino)methyl]-3,5-dimethoxyphenoxy]valeric acid handle; PEG-PS,
polyethylene glycol-polystyrene (graft support); PyAOP, (7-azaben-
zotriazolyloxy)tris(pyrrolidino)phosphonium hexafluorophosphate; Pbf,
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; Pmc, 2,2,5,7,8-
pentamethylchroman-6-sulfonyl; reagent A, TFA-CH2Cl2-dimethyl
sulfide (14:5:1); reagent R, TFA-thioanisole-1,2-ethanedithiol-anisole
(90:5:3:2); Tmob, 2,4,6-trimethoxybenzyl; TFA, trifluoroacetic acid;
XAL, xanthenylamide, the title handles of this paper.
(2) Taken in part from the Ph.D. theses of Y. H., University of
Minnesota, 1996, and M. C. M., University of Minnesota, 1994.
(3) Various portions of this work were reported in preliminary form
at a variety of venues: (a) Barany, G.; Albericio, F. In Peptides 1990:
Proceedings of the Twenty-First European Peptide Symposium; Giralt,
E., Andreu, D., Eds.; ESCOM Science Publishers: Leiden, The
Netherlands, 1991; pp 139-142. (b) Bontems, R. J .; Hegyes, P.;
Bontems, S. L.; Albericio, F.; Barany, G. In Peptides-Chemistry and
Biology: Proceedings of the Twelfth American Peptide Symposium;
Smith, J . A., Rivier, J . E., Eds.; ESCOM: Leiden, The Netherlands
1992; pp 601-602. (c) Han, Y.; Munson, M. C.; Albericio, F.; Barany,
G. In Peptides-Biology and Chemistry: Proceedings of the 1992 Chinese
Peptide Symposium; Du, Y.-c., Tam, J . P., Zhang,Y.-s., Eds.; ESCOM
Science Publishers: Leiden, The Netherlands, 1993; pp 301-304. (d)
Han Y.; Barany, G. In Innovation and Perspectives in Solid Phase
Synthesis & Complementary Technologies: Biological & Biomedical
Application; Epton, R., Ed.; Mayflower Worldwide Ltd.: Birmingham,
England, 1994; pp 525-526. (e) Kates, S. A.; Minor, C. A.; Albericio,
F.; Han, Y.; Barany, G. In Peptides 1994: Proceedings of the Twenty-
Third European Peptide Symposium; Maia, H., Ed.; ESCOM Science
Publishers: Leiden, The Netherlands, 1995; pp 262-264.
(4) (a) University of Minnesota. (b) PerSeptive Biosystems Biosearch
Products. (c) University of Barcelona.
(5) Present addresses: (a) National Starch and Chemical Company,
10 Finderne Avenue, Bridgewater, NJ 08807. (b) Attila J o´zsef Uni-
versity, Department of Organic Chemistry, H-6720 Szeged, Do´m te´r
8, Hungary. (c) Amgen, Inc., 4765 Walnut Street, Boulder, CO 80301.
(d) Department of Chemistry, Brandeis University, Waltham, MA
02254.
(6) (a) Albericio, F.; Barany, G. Int. J . Pept. Protein Res. 1987, 30,
206-216. (b) Albericio, F.; Kneib-Cordonier, N.; Biancalana, S.; Gera,
L.; Masada, R. I.; Hudson, D.; Barany, G. J . Org. Chem. 1990, 55,
3730-3743. (c) Sharma, S. K.; Songster, M. F.; Colpitts, T. L.; Hegyes,
P.; Barany, G.; Castellino, F. J . J . Org. Chem. 1993, 58, 4993-4996.
(d) Albericio, F.; Barany, G. Int. J . Pept. Protein Res. 1993, 41, 307-
312 and references cited in all of these papers.
(7) Hammer, R. P.; Albericio, F.; Gera, L.; Barany, G. Int. J . Pept.
Protein Res. 1990, 36, 31-45 and references cited therein.
(8) Handles (or resin supports) directed toward peptide amides are
reviewed thoroughly in ref 6b and include those developed by the
following: (a) Sieber, P. Tetrahedron Lett. 1987, 28, 2107-2110. (b)
Rink, H. Tetrahedron Lett. 1987, 28, 3787-3790. (c) Stu¨ber, W.; Knolle,
J .; Breiphol, G. Int. J . Pept. Protein Res. 1989, 34, 215-221. More
recent developments were described by the following: (d) Patek, M.;
Lebl, M. Tetrahedron Lett. 1991, 32, 3891-3894. (e) Echner, H.; Voelter
W. In Peptide Chemistry 1992: Proceedings of the 2nd J apan Sympo-
sium on Peptide Chemistry; Yanaihara, N., Ed.; ESCOM Science
Publishers: Leiden, The Netherlands, 1993; pp 113-115. (f) Chao, H.-
G.; Bernatowicz, M. S.; Matsueda, G. R. J . Org. Chem. 1993, 58, 2640-
2644. (g) Pinori, M.; Di Gregorio, G.; Mascagni, P. In Innovation and
Perspectives in Solid Phase Synthesis & Complementary Technologies:
Biological
& Biomedical Application; Epton, R., Ed.; Mayflower
Worldwide Ltd.: Birmingham, England, 1994; pp 635-638. (h) Noda,
M.; Yamaguchi, M.; Ando, E.; Takeda, K.; Nokihara, K. J . Org. Chem.
1994, 59, 7968-7975. (i) Calmes, M.; Daunis, J .; David, D.; J acquier,
R. Int. J . Pept. Protein Res. 1994, 44, 58-60. (j) Ajayaghosh, A.; Pillai,
V. N. R. Tetrahedron Lett. 1995, 36, 777-780. (k) Chan, W. C.; White,
P. D.; Beythlen, J .; Steinauer, R. J . Chem. Soc., Chem. Commun. 1995,
589-592. (l) Holmes, C. P.; J ones, D. G. J . Org. Chem. 1995, 60, 2318-
2319.
S0022-3263(96)00312-X CCC: $12.00 © 1996 American Chemical Society

Xanthenylamide Handles for Synthesis of Peptide Amides
J . Org. Chem., Vol. 61, No. 18, 1996 6327
for chain elongation. Subsequently, cleavage of com-
pleted peptide chains containing the proper amide ter-
minus is achieved by treatment with acid in the presence
of suitable scavengers,8a-f,h,i,k or by photolysis in an
appropriate solvent milieu.7,8j,l,12 Particular success has
been achieved with the tris(alkoxy)benzylamide (PAL)
linkage,6 which is established with the 5-[4-[(Fmoc-
amino)methyl]-3,5-dimethoxyphenoxy]valeric acid handle
(1) and is cleaved efficiently in the presence of trifluoro-
acetic acid (TFA) [∼90% (v/v)].
prepared and evaluated. Cleavage of XAL handles to
provide C-terminal peptide amides occurs at TFA con-
centrations [∼1 to 25% (v/v)] which are lower than those
required to effect cleavage of PAL. This property makes
possible the direct preparation of protected peptide
amides, as well as the synthesis of acid-sensitive peptide
conjugates [illustrated herein for a sulfated peptide,16
CCK-8]. Furthermore, a previously described tryptophan
alkylation problem with PAL supports6b has been shown
to be negligible for sensitive peptide amides synthesized
on XAL supports. Finally, the compatibility of XAL
chemistry with on-resin manipulation of cysteine resi-
dues has been delineated.
The present paper reports on the preparation and
applications of several [[(Fmoc-amino)xanthenyl]oxy]-
alkanoic acids (2a ,b and 3a ,b), which are handle variants
(termed XAL) of the acid-labile xanthenyl protecting
group for carboxamides.13-15 In order to determine the
optimal XAL handle, derivatives with different side-chain
lengths (1 and 4 methylene groups, corresponding to
acetic and valeric acids) and different substituent position
(2 and 3, meta and para) to the xanthenyl system were
Resu lts a n d Discu ssion
P r ep a r a tion of XAL Ha n d les. The required handles
were prepared by efficient four-step routes (overall yield
50-66%), based in part on reasonable literature prece-
dents and featuring direct transformations without sepa-
rate purification steps for intermediates (Schemes 1 and
2). The respective starting materials, 2- or 3-hydroxy-
xanthone (4, 5),17 were obtained in two steps from
o-anisoyl chloride plus 1,4- or 1,3-dimethoxybenzene,
through the corresponding trimethoxybenzophenone
intermediates (6, 7). Alternatively, 4 was obtained by a
new method in a single direct step from commercially
available 2,2′-dihydroxy-4-methoxybenzophenone. The
phenolic function of 4 or 5 was alkylated with ethyl
bromoacetate (series a ) or ethyl 5-bromovalerate (series
b) to provide esters (8, 9) which were saponified to the
corresponding acids (10, 11).15,18 In the 2-XAL series, the
carbonyls of 10a ,b were next reduced selectively with
sodium borohydride in aqueous media at pH 8, to give
xanthydrols 12a ,b. However, the same reactions in the
3-XAL series gave over-reduction to the corresponding
(xanthen-3-yloxy)alkanoic acids 13a ,b, so the critical
reduction step was carried out instead with Zn in the
presence of NaOH, or with sodium amalgam. Regardless
of how they were generated, xanthydrols were trapped
under appropriate acidic conditions with 9-fluorenyl-
methyl carbamate (Fmoc-NH2) (14)8c to give the title
(9) Reviews: (a) Barany, G.; Kneib-Cordonier, N.; Mullen, D. G. Int.
J . Pept. Protein Res. 1987, 30, 705-739. (b) Atherton, E.; Sheppard,
R. C. Solid Phase Peptide Synthesis, A Practical Approach; IRL Press
at Oxford University Press: Oxford, U.K., 1989. (c) Fields, G. B.; Noble,
R. L. Int. J . Pept. Protein Res. 1990, 35, 161-214. (d) Fields, G. B.;
Tian, Z.; Barany, G. In Synthetic Peptides. A User’s Guide; Grant, G.
A., Ed.; W. H. Freeman and Co.: New York, 1992; pp 77-183.
(10) Handles are defined as bifunctional spacers which serve to
attach the initial residue to the polymeric support in two discrete steps.
One end of the handle incorporates features of a smoothly cleavable
protecting group, and the other end allows facile coupling to a
previously functionalized support. For reviews, see refs 9a, 9d, and
the following: (a) Lloyd-Williams, P.; Albericio, F.; Giralt, E. Tetra-
hedron 1993, 49, 11065-11133. (b) Barany, G.; Albericio, F. In
Peptides-Chemistry, Structure and Biology: Proceedings of the Thir-
teenth American Peptide Symposium; Hodges, R. S., Smith, J . A., Eds.;
ESCOM: Leiden, The Netherlands 1994; pp 1078-1080.
(11) For an excellent discussion of possible scavengers, see: King,
D.; Fields, C.; Fields, G. Int. J . Pept. Protein Res. 1990, 36, 255-266.
Further important information about scavengers appears in refs 6b
and 28.
(12) Rich, D. H.; Gurwara, S. K. Tetrahedron Lett. 1975, 301-304.
(13) (a) Shimonishi, Y.; Sakakibara, S.; Akabori, S. Bull. Chem. Soc.
J pn. 1962, 35, 1966-1970. (b) Dorman, L. C.; Nelson, D. A.; Chow, R.
C. L. In Progress in Peptide Research; Lande, S., Ed.; Gordon and
Breach: New York, 1972; Vol. 2, pp 65-68. (c) Sole´, N. A.; Han, Y.;
Va´gner, J .; Gross, C. M.; Tejbrant, J .; Barany, G. In Peptides-
Chemistry, Structure and Biology: Proceedings of the Fourteenth
American Peptide Symposium; Kaumaya, P. T. P., Hodges, R. S., Eds.;
Mayflower Scientific Ltd.: Kingswinford, England, 1996; pp 113-114.
(14) Sieber (ref 8a) has reported on a variant of XAL in which
Merrifield chloromethyl-resin is alkylated by 3-hydroxyxanthone (5),
followed by on-resin reduction by LiBH4 in refluxing THF and acid-
catalyzed trapping with Fmoc-NH2 (14). While superficially similar
to our chemistry for handle 3a (Scheme 2), we had great difficulty
reproducing the published on-resin procedures. Apparently, these
difficulties have now been brought under control with a resin reported
on by Chan et al. (ref 8k).
(15) Our initial studies (refs 3a,b) involved handles 2a and 2b, and
we were unable to prepare 3a . Subsequently, we became aware of the
work of Caciagli, V.; Longobardi, M. G.; Pessi, A. Italian Patent
Application, 19697A, March 16, 1990, SCLAVO SpA, Siena, Italy.
These researchers introduced a Na/Hg reduction step to convert 11a
to 12a [they noted, as do we, difficulties when borohydrides were used
for reduction] and continued successfully to obtain 3a which was then
applied for the solid-phase synthesis of C-terminal amides. They also
reported an unusual side reaction during the alkylation of 5 directly
with chloroacetic acid; besides the desired 11a , the isomer shown below
was formed from reaction of the 9-OH, 3-oxo tautomer of 5.
(16) As reviewed in ref 9d, synthesis of Tyr sulfate-containing
peptides is difficult, due to the substantial acid lability of the sulfate
ester.
(17) Routes to the various isomeric hydroxyxanthones were first
reported in the late 19th/early 20th century German literature, and
some of these compounds have also been isolated as natural products.
For a summary, see: (a) Dictionary of Organic Compounds, H-03557
through H-03560; Chapman & Hall: New York, 1982; Vol. 3, 5th ed.;
pp 3265-3266. More recent, relevant articles include the following:
(b) Van Allan, J . A. J . Org. Chem. 1958, 23, 1679-1682. (c) Royer, R.;
Lechartier, J . P.; Demerseman, P. Bull. Soc. Chim. Fr. 1971, 1707-
1710. For the present work, the procedure developed in ref 17b for
3-hydroxyxanthone (5) was modified and further optimized to prepare
both 5 and 2-hydroxyxanthone (4) in more convenient ways (compare
to ref 17c, which has sketchy experimental details).
(18) Some of these esters and acids are known, see ref 15 and (a)
Cassella Farbwerke Mainkur A.-G. Brit. Pat. 923,132, Apr 10, 1963;
Ger. Appl., J an 20, 1960 [Chem. Abstr. 1963, 59, p9994b]. (b) Puranik,
G. S.; Agasimundin, Y. S.; Rajagopal, S. Indian J . Appl. Chem. 1968,
31, 105-107. (c) Somlai, C.; Nyerges, L.; Penke, B.; Hegyes, P.; Voelter,
W. J . Prakt. Chem./ Chem.-Ztg. 1994, 336, 429-433.

6328 J . Org. Chem., Vol. 61, No. 18, 1996
Han et al.
Sch em e 1
Sch em e 2
compounds 2a ,b and 3a ,b. An alternative route, most
practical in the 2-XAL series,19 transformed 10b to the
corresponding amino derivative 15, which was converted
to the final handle 2b by reaction with N-(Fmoc-oxy)-
succinimide (Fmoc-OSu).20
duced readily onto a range of amino-functionalized
supports. Quantitative anchoring was mediated conve-
niently by the phosphonium and uronium salt deriva-
tives21 of 1-hydroxybenzotriazole (HOBt) or 7-aza-1-
hydroxybenzotriazole (HOAt), in the presence of
N-methylmorpholine (NMM) or N,N-diisopropylethyl-
amine (DIEA), with N,N-dimethylformamide (DMF) as
Use of XAL Ha n d les in Solid -P h a se P ep tid e Syn -
th esis. Handle derivatives 2a ,b and 3a ,b were intro-

Xanthenylamide Handles for Synthesis of Peptide Amides
J . Org. Chem., Vol. 61, No. 18, 1996 6329
F igu r e 1. (A) Kinetics of cleavage for four H-Leu-Ala-Gly-Val-XAL-Phe-PEG-PS supports by TFA-CH2Cl2 (1:19) at 25 °C. (B)
Kinetics of cleavage of H-Leu-Ala-Gly-Val-3-XAL4-Phe-PEG-PS as a function of TFA concentration.
solvent. Parent supports used included standard poly-
styrene (PS) resins, as well as polyethylene glycol-
polystyrene (PEG-PS) graft supports.22 The Fmoc group
was removed from XAL handle resins, and stepwise
elaboration of peptide chains by Fmoc chemistry pro-
ceeded normally according to standard manual or auto-
mated protocols.6b,9d,23
The labilities of various XAL anchors to acid were
assessed by model kinetic studies after assemblies of the
Merrifield tetrapeptide (Leu-Ala-Gly-Val).24 Cleavage by
TFA-CH2Cl2 (1:19) was most efficient for 3-XAL4 (3b:
87% cleavage in 1 h). The 3-XAL1 (3a ) anchor with a
shorter spacer (n ) 1) was cleaved 2-fold more slowly,
and cleavage of 2-XAL (2a ,b) derivatives was slower by
an order of magnitude (Figure 1A). The most labile
linkage, derived from 3-XAL4 (3b), was cleaved even with
a very low concentration of TFA (1%); moderate yields
were observed (∼40%; see Figure 1B).
The kinetic studies suggest three alternative ways in
which XAL-anchored peptides can be released, depending
on the synthetic objective. To obtain protected peptide
amides, low concentrations of TFA are used (see next
section), and when free unprotected peptide amides are
required, the cleavage/deprotection is carried out either
in a two-stage process (“low/high” acid) or in a single step
at higher TFA concentration (“high” acid; illustrations
follow).
(19) This alternative route for the 3-XAL series led to the desired
amine derivative, but it remained contaminated with the xanthydrol
intermediate. Acylation of the amine component in these product
mixtures with Fmoc-OSu provided final XAL handles (3a ,b) which
were difficult to obtain absolutely free of xanthydrol or its decomposi-
tion products.
(20) Ten Kortenaar, P. B. W.; van Dijk, B. G.; Peters, J . M.; Raaben,
B. J .; Adams, P. J . H. M.; Tesser, G. I. Int. J . Pept. Protein Res. 1986,
27, 398-400.
(21) The development and applications of the relevant coupling
reagents are reported by the following: (a) Castro, B.; Dormoy, J . R.;
Evin, G.; Selve, C. Tetrahedron Lett. 1975, 1219-1222. (b) Dourtoglou,
V.; Ziegler, J .-C.; Gross, B. Tetrahedron Lett. 1978, 1269-1272. (c)
Hudson, D. J . Org. Chem. 1988, 53, 617-624. (d) Fournier, A.; Wang,
C. T.; Felix, A. M. Int. J . Pept. Protein Res. 1988, 31, 86-97. (e) Knorr,
R.; Trzeciak, A.; Bannwarth, W.; Gillessen, D. Tetrahedron Lett. 1989,
30, 1927-1930. (f) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397-
4398. (g) Carpino, L. A.; El-Fahan, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201-203. (h) Kates, S. A.; Minor,
C. A.; Shroff, H.; Haaseth, R. C.; Triolo, S.; El-Faham, A.; Carpino, L.
A.; Albericio, F. In Peptides 1994: Proceedings of the Twenty-Third
European Peptide Symposium; Maia, H. L. S., Ed.; ESCOM: Science
Publishers: Leiden, The Netherlands, 1995; pp 248-249.
(22) (a) Barany, G.; Albericio, F.; Sole´, N. A.; Griffin,, G. W.; Kates,
S. A.; Hudson, D. In Peptides 1992: Proceedings of the Twenty-Second
European Peptide Symposium; Schneider C. H., Eberle, A. N., Eds.;
ESCOM Science Publishers: Leiden, The Netherlands, 1993; pp 267-
268. (b) Zalipsky, S.; Chang, J . L.; Albericio, F.; Barany, G. React.
Polym. 1994, 22, 243-258 and references cited therein.
P r ep a r a tion of P r otected P ep tid e Am id es w ith
3-XAL. When TFA-CH2Cl2 (1:99) is used as the cleav-
age cocktail, tert-butyl-type protecting groups that are
typical for Fmoc SPPS are not removed. However, the
aforementioned dilute acid reagent is not strong enough
to promote full cleavage of the most labile XAL anchor,
i.e., that derived from 3-XAL4 (3b), either (see Figure 1B).
The unsatisfactory saturation of cleavage yield is appar-
ently due to buffering of TFA by amide bonds and other
functional groups in peptides.25 To overcome this prob-
lem, we employ repeated short pulses with TFA-
CH2Cl2 (1:99). The protected peptide amide Fmoc-Ala-
Pro-Trp-Ala-Val-Leu-Glu(OtBu)-Val-Ala-NH2 was ob-
tained in 84% yield and excellent purity (>90%) after six
consecutive 5 min treatments with TFA-CH2Cl2 (1:99)
(Figure 2).
P r ep a r a tion of Un p r otected P ep tid e Am id es w ith
XAL. In a two-stage process, initial treatment in the
presence of 1-25% TFA released peptides in high yields,
(23) (a) Barany, G.; Gross, C. M.; Ferrer, M.; Barbar, E.; Pan, H.;
Woodward, C. In Techniques in Protein Chemistry VII; Marshak, D.
R., Ed.; Academic Press: New York, 1996; pp 503-514. (b) Kates, S.
A.; Diekmann, E.; El-Faham, A.; Herman, L. W.; Ionescu, D.; McGuin-
ness, B. F.; Triolo, S. A.; Albericio, F.; Carpino, L. A. In Techniques in
Protein Chemistry VII; Marshak, D. R., Ed.; Academic Press: New
York, 1996; pp 515-523.
(24) (a) Merrifield, R. B. J . Am. Chem. Soc. 1963, 85, 2149-2154.
(b) Kent, S. B. H.; Mitchell, A. R.; Barany, G.; Merrifield, R. B. Anal.
Chem. 1978, 50, 155-159.
(25) This “buffering” phenomenon was first discovered and ad-
dressed by Riniker, B.; Flo¨rsheimer, A.; Fretz, H.; Sieber, P.; Kamber,
B. Tetrahedron 1993, 49, 9307-9320.

6330 J . Org. Chem., Vol. 61, No. 18, 1996
Han et al.
F igu r e 2. Analytical HPLC of crude Fmoc-Ala-Pro-Trp-Ala-
Val-Leu-Glu(OtBu)-Val-Ala-NH2 prepared starting with Fmoc-
3-XAL4-PEG-PS (derived from handle 3b; further details in
the Experimental Section). HPLC was performed on a Delta
Pak C18 reversed-phase column (3.9 × 150 mm, 5 µm), with a
linear gradient using 0.036% TFA in CH3CN and 0.045%
aqueous TFA, from 1:9 to 1:0 over 20 min, flow rate 1.0
mL/min.
F igu r e 3. Analytical HPLC of crude prothrombin fragment
(1-9), H-Ala-Asn-Lys-Gly-Phe-Leu-Glu-Glu-Val-NH2 prepared
on Fmoc-3-XAL4-PEG-PS support (0.21 mmol/g, derived from
handle 3b), after peptide chain assembly and cleavage with
reagent R for 2 h at 25 °C. Couplings of appropriately protected
Fmoc-amino acid derivatives were performed automatically on
a continuous-flow PerSeptive Biosystems 9050 peptide syn-
thesizer, using an HATU/DIEA protocol (Table 2). HPLC was
performed as for Figure 2, except the gradient was over 30
min.
but with partial retention of side-chain tert-butyl protec-
tion (e.g., see Figure 5A later). Therefore, a second step
followed in which the acid (TFA) concentration was
increased to an overall level of 50%, so that full depro-
tection was achieved in solution.26 Similar overall yields
and purities of free C-terminal peptide amides were also
observed when higher acid concentrations, as usually
applied at the end of standard Fmoc syntheses, were used
for direct cleavage/deprotection in a single step. This
approach was demonstrated by syntheses of fragment
1-9 of prothrombin [Figure 3: anchor derived from
3-XAL4 (3b), 97% cleavage with reagent R, TFA-thio-
anisole-1,2-ethanedithiol-anisole (90:5:3:2), 2 h, 25 °C]
and the (65-74) fragment27 of the acyl carrier protein
[Figure 4: anchor derived from 3-XAL4 (3b), 95% cleav-
age with reagent R].
P r ep a r a tion of Tr yp top h a n -con ta in in g P ep tid es
w ith XAL.28 The sequence pGlu-Leu-Thr-Phe-Thr-Pro-
Gly-Trp-NH2, derived from Tabanus atratus adipokinetic
hormone,29 was developed previously as a sensitive and
quantifiable model for a tryptophan alkylation side
reaction with PAL supports.6b Cleavage of peptide-PAL-
Nle-MBHA-PS with reagent R for 2 + 8 h proceeded in
only ∼55% yield, with the uncleaved peptide presumed
to be linked to resin-bound PAL via the 2-position of the
indole side chain. Alternatively, cleavage of peptide-PAL-
F igu r e 4. Analytical HPLC of crude acyl carrier protein (65-
74), H-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH2 prepared
on Fmoc-3-XAL4-PEG-PS support (derived from handle 3b).
All other details identical to those of Figure 3. The slight
shoulder just before the main peak is the des(Val) species,
whereas the des(Asn) species elutes just after the main peak.
(26) To obtain Arg-containing free peptides that were originally
protected with either Pmc or Pbf, it is necessary to remove CH2Cl2
and add neat TFA in the presence of scavengers.
(27) The acyl carrier protein (65-74) decapeptide is considered a
“difficult” sequence for stepwise chain assembly and was described first
by Hancock, W. S.; Prescott, D. J .; Vagelos, P. R.; Marshall, G. R. J .
Org. Chem. 1973, 38, 774-781.
(28) For a systematic study of cleavage conditions and a comparison
of the relative utility of 2-XAL4 vs PAL supports for the preparation
of indolicidin [a 13-residue peptide amide with five Trp residues], see:
Van Abel, R. J .; Tang, Y.-Q.; Rao, V. S. V.; Dobb, C. H.; Tran, D.;
Barany, G.; Selsted, M. E. Int. J . Pept. Protein Res. 1995, 45, 401-
409.
(29) J affe, H.; Raina, A. K.; Riley, C. T.; Fraser, B. A.; Nachman, R.
J .; Vogel, V. W.; Zhang, Y. S.; Hayes, T. K. Proc. Nat. Acad. Sci. U.S.A.
1989, 86, 8161-8164.
MBHA-PS (omitting the intervening Nle residue which
provides an acid-stable point of linkage) with reagent R
or a variety of other acid reagent/scavenger cocktails
resulted in not only the desired peptide but also sub-
stantial amounts (nearly equimolar, and often higher) of
the tryptophan-alkylated byproduct being released into
solution. For these experiments, the byproduct, which
included a PAL-NH2 moiety, was characterized by NMR
and FABMS. In contrast to the just-mentioned results
with PAL, the present paper shows that cleavage of
peptide-2-XAL4-Nle-MBHA-PS (derived from handle 2b)

Xanthenylamide Handles for Synthesis of Peptide Amides
J . Org. Chem., Vol. 61, No. 18, 1996 6331
Ta ble 1. Acid olytic Clea va ge of P AL a n d XAL An ch or in g
Lin k a ges for P ep tid es w ith C-Ter m in a l Tr yp top h a n a
cleavage yield,e %
entry
peptideb,c
cleavage cocktaild
PAL
XAL
1
2
3
4
5
6
7
H-SSIPDAFW-NH2
H-SSIPDAFWW-NH2 TFA-CH2Cl2 (9:1)
TFA-CH2Cl2 (9:1)
14
33
89
92
96
95
91
96
94
H-SSIPDAFW-NH2
H-SSIPDAFW-NH2
H-SSIPDAFW-NH2
reagent Rd
TFA-H2O (9:1)
TFA-CH2Cl2 (2:3)
H-SSIPDAFWW-NH2 reagent Rd
H-SSIPDAFWW-NH2 TFA-H2O (9:1)
a
The rationale of these studies is explained in the text.
Peptides were assembled on Fmoc-3-XAL4-PEG-PS (0.21 mmol/
b
g; derived from handle 3b). Couplings of appropriately protected
Fmoc-amino acid derivatives were performed automatically on a
continuous-flow PerSeptive Biosystems 9050 peptide synthesizer,
using an HATU/DIEA protocol (Table 2). All cleavages were
carried out for 1 h at 25 °C, using the cleavage cocktail in column
d
3. c Representative analytical HPLC data shown in Figure 6. All
cleavage cocktails were prepared freshly immediately before use.
Reagent R is TFA-thioanisole-1,2-ethanedithiol-anisole (90:5:
3:2). e Cleavage yields were calculated on the basis of amino acid
analyses of peptide-resins before and after the cleavage, with
respect to norleucine serving as “internal reference” amino acid.
or alkylation-sensitive residues (Trp, Met), as well as the
acid-labile sulfate on tyrosine. In the present work,
Fmoc-3-XAL1-PEG-PS (derived from handle 3a ) was used
as the starting point to assemble the linear sequence by
standard Fmoc chemistry. Incorporation of the tyrosine
sulfate residue, as its NR-Fmoc, side-chain barium salt,
was accomplished directly by a BOP/HOBt/NMM cou-
pling. The two aspartate residues were protected as allyl
esters and deprotected upon completion of chain assembly
by use of Pd(PPh3)4 in CHCl3-HOAc-NMM (20:1:0.5),
for 2 h at 25 °C.32 The overall cleavage yield, using TFA-
CH2Cl2-Η2Ã (1:18:1) for 15 min at 25 °C, was 71%, and
the desired product represented >95% of the total peptide
material (Figure 7A). This strategy represented a clear
improvement over our previous result3b starting with
Fmoc-2-XAL4-PEG-PS (derived from handle 2b) and
using tBu ester protection for the two aspartate residues.
A number of cleavage conditions were examined, and
TFA-CH2Cl2-â-mercaptoethanol-anisole (50:45:3:2), 15
min, 25 °C, was found to represent the optimal compro-
mise between acid-promoted loss of sulfate and incom-
plete deprotection. The overall cleavage yield was 90%,
and the desired product represented ∼70% of the total
peptide material (Figure 7B). Also observed from this
earlier work were 10% of unsulfated CCK-8 and 20% of
CCK-8 sulfate derivatives with one or the other of the
aspartates retaining tert-butyl side-chain protection.
F igu r e 5. Analytical HPLC of crude samples of T. atratus
adipokinetic hormone (ref 29) prepared on Fmoc-2-XAL4-PEG-
PS support (derived from handle 2b; further details in the
Experimental Section). HPLC was performed on a Vydac C18
reversed-phase column (4.6 × 250 mm, 5 µm), with a linear
gradient over 20 min, using CH3CN and 0.01 N HCl from 1:9
to 3:2; flow rate 1.2 mL/min. Panel A: sample obtained after
initial cleavage with TFA-CH2Cl2 (1:19) for 2 h at 25 °C. Panel
B: sample obtained after adding neat TFA to bring the overall
filtrate to TFA-CH2Cl2 (1:1) for 2 h at 25 °C.
with reagent R provides the desired peptide in excellent
purity and yield (>90%), meaning that any putative
tryptophan alkylation side reaction would occur at a
relatively low level. Even more striking, the purities and
yields upon cleavage of XAL did not seem to require the
scavengers, individual or in combination, as had been
optimized in the PAL series.6b Thus, release from the
support was achieved adequately with TFA-CH2Cl2
(1:19) for 1 h at 25 °C, and TFA-CH2Cl2 (1:1) for 1 h
further in solution was needed to deblock fully the
identifiable two mono-tert-butyl and single di-tert-butyl
intermediates (Figure 5).
A further demonstration of negligible back-alkylation
in the XAL series was obtained when two fragments of
the protein RHK 130 [H-Ser-Ser-Ile-Pro-Asp-Ala-Phe-Trp-
NH2 (458-465) and H-Ser-Ser-Ile-Pro-Asp-Ala-Phe-Trp-
Trp-NH2 (458-466)] containing one and two residues of
Trp, respectively, at the C-terminal end, were synthe-
sized in parallel on PAL vs 3-XAL4 (derived from handle
3b) derivatized PEG-PS supports. Cleavage of peptide-
resins in the absence of scavengers [TFA-CH2Cl2 (9:1)]
gave in both cases better yields when XAL resins were
used (Table 1, entries 1 and 2). Furthermore, the
presence of scavengers (Table 1, entries 3, 4, 6, 7) did
not improve significantly the cleavage yield, nor did lower
amounts of TFA (Table 1, entry 5) decrease the yields.
HPLC analyses of the crude cleaved peptides showed
better homogeneities when starting with XAL handles
(Figure 6).
P r ep a r a tion of Oxytocin w ith XAL. The target
sequence was of interest as a test of the integration of
the XAL approach with our experiences on thiol protec-
tion and disulfide bond formation.33,34 The linear se-
(31) Adler, G., Beglinger, C., Eds. Cholecystokinin Antagonists in
Gastroenterology; Springer-Verlag: Berlin, 1991.
(32) (a) Albericio, F.; Barany, G.; Fields, G. B.; Hudson, D.; Kates,
S. A.; Lyttle, M. H.; Sole´, N. A. In Peptides 1992: Proceedings of the
Twenty-Second European Peptide Symposium; Schneider C. H., Eberle,
A. N., Eds.; ESCOM Science Publishers: Leiden, The Netherlands,
1993; pp 191-193. (b) Kates, S. A.; Daniels, S. B.; Albericio, F. Anal.
Biochem. 1993, 212, 303-310.
(33) (a) Garc´ıa-Echeverr´ıa, C.; Albericio, F.; Pons, M.; Barany, G.;
Giralt, E. Tetrahedron Lett. 1989, 30, 2441-2444. (b) Albericio, F.;
Hammer, R. P.; Garc´ıa-Echeverr´ıa, C.; Molins, M. A.; Chang, J . L.;
Munson, M. C.; Pons, M.; Giralt, E.; Barany, G. Int. J . Pept. Protein
Res. 1991, 37, 402-413. (c) Review: Andreu, D.; Albericio, F.; Sole´,
N. A.; Munson, M. C.; Ferrer, M.; Barany, G. In Methods in Molecular
Biology, Vol. 35: Peptide Synthesis Protocols; Pennington, M. W., Dunn,
B. M., Eds.; Humana Press: Totowa, NJ , 1994; pp 91-169.
P r ep a r a tion of CCK-8 Su lfa te w ith XAL. The title
sulfated octapeptide31 is recognized to be a challenging
synthetic target due to the presence of several acid- and/
(30) Tamkun, M.; Knoth, E. M.; Walbridge, J . A.; Kroemer, H.;
Roden, D. M.; Glover, D. M. FASEB J . 1991, 5, 331-337.

6332 J . Org. Chem., Vol. 61, No. 18, 1996
Han et al.
F igu r e 6. Analytical HPLC of crude samples of fragments (458-465) [panel A] and (485-466) [panel B] of protein RHK 1
(H-Ser-Ser-Ile-Pro-Asp-Ala-Phe-Trp-NH2 and H-Ser-Ser-Ile-Pro-Asp-Ala-Phe-Trp-Trp-NH2, respectively). Details of the syntheses
are in the footnotes to Table 1, and HPLC was performed as for Figure 3. In each case, syntheses marked with a prime started
with Fmoc-3-XAL4-PEG-PS derived from handle 3b , and syntheses marked with
a
double prime started with
Fmoc-PAL-PEG-PS.
asparagine and glutamine remained unprotected. In
some experiments, final cleavage was achieved with low
concentrations of TFA, i.e., TFA-CH2Cl2-Et3SiH (7:92:
1) for 1 h at 25 °C (95% cleavage yield). Surprisingly,
the expected dihydrooxytocin product was accompanied
by a second, minor (∼25%) peak on HPLC (Figure 8A).
When a higher concentration of TFA was used, i.e., TFA-
CH2Cl2-Et3SiH (70:29:1), the ratio of the two peaks was
inverted (∼5:2) (Figure 8B). The additional peak was
identified by FABMS and dithiothreitol reduction studies
(Figure 8C) as cyclized oxytocin. Thus, high concentra-
tions of TFA seem to favor direct formation of the
disulfide upon XAL cleavage; as the concentration of acid
is decreased, reduced product is favored. Shorter cleav-
age times, e.g., 30 min, resulted in almost exclusive
formation of reduced product even with high levels of
TFA. Such phenomena have not been observed previ-
ously with PAL and are not understood at present.
We also tested oxidative cleavage/cyclization of Cys-
(Tmob) while the protected peptide was still anchored on
the resin. Reagents used were thallium(III) trifluoroac-
etate [1.1-1.3 equiv in DMF-anisole (19:1)], as well as
iodine [3 equiv in TFA-CH2Cl2-anisole (82:15:3)].33 In
most cases, the absolute yields (∼40%) were comparable
to those obtained with PAL-anchored peptides.33b,34 HPLC
traces were remarkably clean (upwards of 90% pure for
crude cleavage, see Figure 9), and cleavage yields were
almost always 90% or higher. Due to the direct formation
of oxytocin as the cyclized disulfide upon cleavage of XAL
anchors at higher TFA concentrations (see previous
paragraph), it is difficult to know to what extent these
good results reflect effective on-resin oxidation.
F igu r e 7. Analytical HPLC of crude samples of CCK-8
sulfated peptide (details of syntheses are in the Experimental
Section). Panel A: starting with Fmoc-3-XAL1-PEG-PS (de-
rived from handle 3a ). HPLC was performed similar to that
described in the legend to Figure 2, except the gradient over
20 min ranged from 1:9 to 2:3. Panel B: starting with Fmoc-
2-XAL4-PEG-PS (derived from handle 2b). HPLC was per-
formed similar to that described in the legend to Figure 5,
except the gradient over 20 min ranged from 1:9 to 2:3 and
the flow rate was 1.0 mL/min.
quence was assembled smoothly on an Fmoc-2-XAL4-Ala-
MBHA support derived from handle 2b (Scheme 3). Our
S-2,4,6-trimethoxybenzyl (Tmob)34a protecting group was
Con clu sion s
t
used to block the â-thiol group of cysteine, while Bu was
This paper has described optimal chemistries for
preparation of several XAL handles. The crucial step is
reduction of a xanthone carbonyl, a transformation which
is prone to side reactions and difficult to achieve quan-
titatively and reproducibly in the solid-phase mode.14,15
Consequently, our preformed handle approach,6,7,10 in
used for the phenol of tyrosine, and side chains of
(34) (a) Munson, M. C.; Garc´ıa-Echeverr´ıa, C.; Albericio, F.; Barany,
G. J . Org. Chem. 1992, 57, 3013-3018. (b) Munson, M.; Lebl, M.;
Slaninova´, J .; Barany, G. Pept. Res. 1993, 6, 155-159. (c) Munson, M.
C.; Barany, G. J . Am. Chem. Soc. 1993, 115, 10203-10216.

Xanthenylamide Handles for Synthesis of Peptide Amides
Sch em e 3
J . Org. Chem., Vol. 61, No. 18, 1996 6333
F igu r e 8. Analytical HPLC of crude samples of oxytocin
prepared starting with Fmoc-2-XAL4-PEG-PS (derived from
handle 2b; further details in the Experimental Section). HPLC
was performed similar to that described in the legend to Figure
5, except the gradient over 20 min ranged from 1:9 to 2:3. Panel
A: sample obtained when a low concentration of TFA was used
[TFA-CH2Cl2-Et3SiH (7:92:1) for 1 h at 25 °C]. Panel B:
sample obtained when a higher concentration of TFA was used
[TFA-CH2Cl2-Et3SiH (70:29:1) for 1 h at 25 °C]. Panel C:
material from sample B was concentrated and redissolved in
0.01 M NaHCO3, pH ∼9, and solid dithiothreitol (∼5-8 equiv)
was added; reduction was carried out for 1 h at 40 °C followed
by chromatography.
F igu r e 9. Analytical HPLC of crude cyclized oxytocin pre-
pared starting with Fmoc-2-XAL4-PEG-PS (derived from handle
2b; further details in the Experimental Section). HPLC was
exactly as in Figure 8.
optimal for the preparation of both free and protected
C-terminal peptide amides. Final cleavage from the
support can be accomplished with dilute TFA (1-5%) in
CH2Cl2. In contradistinction to experiences with other
handles, scavengers such as dimethyl sulfide, 1,2-
ethanedithiol, anisole, and/or thioanisole are not required
for high cleavage yields, nor for the optimal purity of Trp-
containing peptides [note that scavengers are still needed
for removal Pmc,35 Pbf,36 and/or Tmob34 from peptides].
which synthesis, purification, and characterization in
solution precede smooth incorporations onto the support,
is particularly appropriate in the XAL system. Addition-
ally, the availability of handles allows the anchoring
chemistry to be adapted to a wide range of useful solid-
phase synthesis supports.9,22
(35) Ramage, R.; Green, J .; Blake, A. J . Tetrahedron 1991, 47, 6353-
6370.
(36) Carpino, L. A.; Schroff, H.; Triolo, S. A.; Mansour, E.-S. M. E.;
Wenschuh, H.; Albericio, F. Tetrahedron Lett. 1993, 34, 7829-7832.
The 3-XAL4 (3b) handle containing a four-carbon
spacer in position 3 of the xanthenyl ring appears to be

6334 J . Org. Chem., Vol. 61, No. 18, 1996
Han et al.
(120 mL) and combined with solid Fmoc-NH2 (14) (3.3 g, 2
equiv). The brown reaction mixture was allowed to stand at
25 °C for 45 min (product began to precipitate after 15 min)
and concentrated in vacuo to about a third to half of the
original volume. The crude product was collected by filtration
and purified by extraction with boiling EtOAc-MeOH (50 mL,
9:1 v/v). After cooling, the title light-beige solid was collected
by filtration, washed with EtOAc (3 × 20 mL), and dried in
vacuo over NaOH: yield 2.10 g (64%). Meth od B (P r e-
fer r ed ). A mixture of keto acid 11a (1.0 g, 3.7 mmol), NaOH
(solid flakes) (0.74 g, 18 mmol), and activated zinc37 (0.96 g,
15 mmol) was refluxed in 95% EtOH (100 mL) for 6 h to
provide a light-pink solution. Zinc was removed by filtration;
this was followed by washing with hot 95% EtOH (3 × 5 mL),
and the combined filtrates were concentrated to give a light-
pink solid which was dissolved in a mixture of TFA (2.3 mL,
30 mmol) and 1,2-dimethoxyethane (50 mL).38 Fmoc-NH2 (14)
(1.1 g, 4.4 mmol) was added to this yellow mixture in one
portion, and the resultant solution was stirred for 4 h at 25
°C. Solid K2CO3 (1.0 g, 7.4 mmol) was then added, and within
1 h at 25 °C, a white precipitate formed. The resultant
potassium salt was collected by filtration, washed with EtOH
(3 × 10 mL) to remove excess 14, and then suspended in a
mixture of H2O-EtOH (50 mL, 5:1 v/v) and acidified to pH
∼5 by addition of 1 M aqueous KHSO4 (1.6 mL). The resultant
light-beige solid was collected by filtration, washed with H2O
(3 × 10 mL), and dried in vacuo over P2O5: yield 1.32 g (73%);
mp 170-172 °C; Rf[CHCl3-MeOH-HOAc (10:1:0.1)] 0.53; 1H
NMR (CD3SOCD3) δ 8.25 (d, J ) 8.8 Hz, NH), 7.87 (d, J ) 7.2
Hz, 2H), 7.68 (d, J ) 7.2 Hz, 2H), 7.10-7.41 (m, 9H), 6.68 (dd,
J ) 2.5 and 8.5 Hz, 1H), 6.57 (d, J ) 2.5 Hz, 1H), 5.89 (d, J )
8.8 Hz, 1H), 4.50 (s, 2H), 4.37 (d, J ) 6.5 Hz, 2H), 4.24 (t, J )
6.5 Hz, 1H).
Exp er im en ta l Section
Most of the materials and general synthetic and analytical
procedures have been described in our earlier publica-
tions.6,7,10,22,23,32 1H NMR spectra were recorded at 300 MHz
on either a Nicolet NT-300 WB or an IBM NR/300 instrument
and at 200 MHz on an IBM NR/200 instrument, using either
CDCl3 or CD3SOCD3 as solvent. 13C NMR spectra were
obtained at 75 or 50 MHz on the same instruments, and
assignments of carbon resonances were facilitated by DEPT
experiments. Fast atom bombardment mass spectrometry
(FABMS) to characterize synthetic peptides was carried out
on a VG 707E-HF instrument, with glycerol or thioglycerol
matrices being used to obtain both positive and negative ion
spectra. Elemental analyses were performed by M-H-W
Laboratories, Phoenix, AZ.
Thin-layer chromatography was performed on Analtech or
Merck silica gel GF plates (250 µm, 10 × 20 cm), and
compounds were observed by fluorescence quenching and by
spraying with a dilute ethanolic ninhydrin solution. Amino
acid analyses and analytical HPLC were carried out on
previously described Waters and Beckman instruments.
[[9-[(9-F lu or en ylm eth yloxyca r bon yl)a m in o]xa n th en -
2-yl]oxy]a cetic Acid [2-XAL1] (2a ). A solution of Fmoc-NH2
(14) (0.91 g, 3.6 mmol) in glacial acetic acid (HOAc, 30 mL)
was added to a solution of the xanthydrol intermediate 12a
(1.0 g, 3.3 mmol) in HOAc (55 mL). Next a solution of
p-toluenesulfonic acid (0.1 g, 0.5 mmol) in HOAc (10 mL) was
added over 20 min, and the reaction mixture was stirred for
24 h at 25 °C. The product precipitated slowly as a white solid,
which was filtered, washed with H2O (4 × 10 mL), and dried
in vacuo over P2O5: yield 1.34 g (83%); mp 217-218 °C; TLC
pure, Rf[acetone-EtOH-H2O (12:1:1)] 0.54; 1H NMR (CD3-
SOCD3) δ 8.38 (d, J ) 8.8 Hz, NH), 7.89 (d, J ) 7.3 Hz, 2H),
7.72 (d, J ) 7.3 Hz, 2H), 7.35-7.5 (m, 6H), 7.1-7.2 (m, 3H),
6.93 (narrow m, 2H), 5.93 (d, J ) 8.6 Hz, 1H), 4.66 (s, 2H),
4.40 (m, 2H), 4.25 (m, 1H).
Anal. Calcd for C30H23NO6 (493.52): C, 73.00; H, 4.70; N,
2.84. Found: C, 72.98; H, 4.81; N, 2.77.
5-[[9-[(9-Flu or en ylm eth yloxycar bon yl)am in o]xan th en -
3-yl]oxy]va ler ic Acid [3-XAL4] (3b). Meth od A. Com-
pound 3b was prepared essentially as reported for compound
3a , but carried out starting with keto acid 11b (0.50 g, 1.6
mmol). The crude product solid was purified by extraction
with boiling EtOAc-hexane (15 mL, 1:1 v/v), since the use of
EtOAc-MeOH gave a gel which was impossible to filter: yield
0.54 g (63%). Meth od B (P r efer r ed ). Compound 3b was
prepared again as reported for compound 3a , starting with
keto acid 11b (1.0 g, 3.2 mmol): yield 1.33 g (77%); mp 120-
122 °C; Rf[CHCl3-MeOH-HOAc (10:1:0.1)] 0.55; 1H NMR
(CD3SOCD3) δ 8.27 (d, J ) 8.8 Hz, NH), 7.89 (d, J ) 7.2 Hz,
2H), 7.71 (d, J ) 7.2 Hz, 2H), 7.14-7.42 (m, 9H), 6.71-6.76
(m, 2H), 5.89 (d, J ) 8.8 Hz, 1H), 4.41 (d, J ) 6.5 Hz, 2H),
4.24 (t, J ) 6.5 Hz, 1H), 4.01 (t, J ) 5.9 Hz, 2H), 2.31 (t, J )
6.8 Hz, 2H), 1.70 (m, 4H). Method B was carried out on a 0.2
mol scale at Hoffmann-La Roche, Inc., with comparable yields
and purities.
Anal. Calcd for C30H23NO6 (493.52): C, 73.00; H, 4.70; N,
2.84. Found: C, 73.06; H, 4.95; N, 3.01.
5-[[9-[(9-Flu or en ylm eth yloxycar bon yl)am in o]xan th en -
2-yl]oxy]va ler ic Acid [2-XAL4] (2b). Meth od A. A suspen-
sion of xanthydrol intermediate 12b (2.05 g, 6.5 mmol) and
Fmoc-NH2 (14) (1.46 g, 6.5 mmol) in glacial HOAc (50 mL)
was heated to 50 °C, providing a clear yellow solution.
A
cottonlike white solid started to form after 10 min; the reaction
was maintained for 1 h at 50 °C, and then the mixture was
cooled to 25 °C. The light-beige product was collected by
filtration, washed with H2O (3 × 20 mL) and ethyl ether (3 ×
20 mL), and dried in vacuo over P2O5 at 25 °C: yield 2.30 g
(68%). Meth od B. Xanthydrylamine 15 (1.0 g, 3.2 mmol) was
suspended in H2O (100 mL), and Et3N (0.82 g, 1.13 mL, 8.1
mmol, 2.5 equiv) was added to furnish a clear yellow solution.
A solution of Fmoc-OSu (1.3 g, 3.85 mmol, 1.2 equiv) in
CH3CN (70 mL) was then added, and a gel-like precipitate
started to form after 30 min. The reaction was maintained
for a total of 3 h at 25 °C, and then HOAc was added to reach
pH 3.3. The light-beige product was collected by filtration,
washed with H2O (3 × 50 mL) and CH3CN (3 × 50 mL), and
dried in vacuo over P2O5 at 25 °C: yield 1.55 g (91%); mp 210-
211 °C; Rf[EtOAc-MeOH (5:1)] 0.53 (single spot); 1H NMR
(CD3SOCD3) δ 8.36 (d, J ) 8.8 Hz, NH), 7.89 (d, J ) 7.2 Hz,
2H), 7.71 (d, J ) 7.2 Hz, 2H), 7.35-7.5 (m, 6H), 7.1-7.2 (m,
3H), 6.9-7.0 (m, 2H), 5.92 (d, J ) 8.8 Hz, 1H), 4.42 (d, J ) 6.8
Hz, 2H), 4.26 (t, J ) 6.8 Hz, 1H), 3.93 (br t, 2H), 2.29 (t, J )
7.0 Hz, 2H), 1.6-1.8 (m, 4H).
Anal. Calcd for C33H29NO6 (535.60): C, 73.99; H, 5.46; N,
2.62. Found: C, 73.80; H, 5.36; N, 2.69.
2-Hyd r oxyxa n th on e (4). A mixture of crude 2,2′,5-tri-
methoxybenzophenone (6) (32 g, 118 mmol) and pyridine
(37) It is strongly recommended to use freshly activated zinc,
prepared as follows: zinc dust (2.0 g) was covered with 1.5 N aqueous
HCl (20 mL) for ∼5 min and then washed successively with H2O (3 ×
10 mL), EtOH (3 × 10 mL), acetone (3 × 5 mL), and ethyl ether (3 ×
5 mL). The subsequent reaction was noted to fail sometimes when the
zinc had been stored more than a day in a loosely sealed bottle, or
when the reaction solvent was absolute EtOH instead of 95% EtOH
as in the text experimental description.
(38) When the same reaction sequences were carried out using as
solvent neat HOAc (compare to procedures for 2a and 2b) instead of
DME containing TFA, the desired handles 3a and 3b could not be
isolated. Rather, two phenomena took place: (i) the unstable inter-
mediate xanthydrol (never isolated) disproportionated to starting
ketone 11a or 11b and corresponding over-reduced xanthene 13a or
13b and (ii) Fmoc-NH2 (14) was converted to dibenzofulvene
Rf[hexane-EtOAc (10:1)] 0.56; 1H NMR (CDCl3) δ 7.74 (d, J ) 7.2 Hz,
2H), 7.70 (d, J ) 7.5 Hz, 2H), 7.38 (td, J ) 0.9 and 7.2 Hz, 2H), 7.30
(td, J ) 1.2 and 7.5 Hz, 2H), 6.08 (s, 2H) [matched signals from
treatment of Fmoc-glycine with NH3 in MeOH, quick evaporation, and
redissolving in CDCl3]; EIMS (solid probe, 70 eV, 25 °C) m/z 178.1
(calcd for C14H10, 178.08)].
Anal. Calcd for C33H29NO6 (535.60): C, 74.00; H, 5.45; N,
2.61. Found: C, 73.85; H, 5.46; N, 2.45.
[[9-[(9-F lu or en ylm eth yloxyca r bon yl)a m in o]xa n th en -
3-yl]oxy]a cetic Acid [3-XAL1] (3a ). Meth od A. Following
Caciagli et al.,15 an amalgam was made from cut sodium (1.2
g, 53 mmol) and mercury (7.5 mL, 0.5 mol) which were warmed
to 75 °C for 1 h. A suspension of keto acid 11a (2.0 g, 7.4
mmol) in 95% EtOH (160 mL) was then added, and the white
reaction mixture was heated to reflux for 2 h. The mixture
was filtered while hot to remove residual amalgam and then
concentrated to give a white solid which was dissolved in HOAc

Xanthenylamide Handles for Synthesis of Peptide Amides
J . Org. Chem., Vol. 61, No. 18, 1996 6335
hydrochloride (172 g, 1.5 mol) was heated to a gentle reflux
(210 °C bath) under N2. After 10 h of reaction, the mixture
was quenched by pouring into ice H2O (400 g), and the
resultant yellow-green precipitate was collected, washed with
H2O (2 × 100 mL) and 6 N aqueous HCl (3 × 100 mL), and
combined with hot 95% EtOH (50 mL) to form a suspension.
The solid was collected again once the solvent had cooled,
washed with 95% EtOH (2 × 25 mL), and dried in vacuo:
yield: 20.6 g (82%); mp 245-246 °C [lit.17a mp 241 °C];
washed with acetone (3 × 50 mL) and ether (3 × 50 mL),
concentrated, and placed under hexane, whereupon a powdery
brown solid formed: yield 16.1 g (82%); mp 91-92 °C;
Rf[benzene-EtOH (20:3)] 0.81 (one spot); 1H NMR (CD3-
SOCD3) δ 8.19 (dd, J ) 1.6 and 7.9 Hz, 1H), 7.86 (td, J ) 1.7
and 7.8 Hz, 1H), 7.42-7.67 (m, 5H), 4.94 (s, 2H), 4.20 (q, J )
7.1 Hz, 2H), 1.32 (t, J ) 7.1 Hz, 3H). An analytical sample
from n-hexane-EtOH (1:1) was fine light-beige needles, mp
96-98 °C [lit.15,18a mp 97-98 °C].
1
Rf[benzene-EtOH (20:3)] 0.54; H NMR (CD3SOCD3) δ 10.0
Anal. Calcd for C17H14O5 (298.28): C, 68.45; H, 4.73.
Found: C, 68.65; H, 5.01.
(1H, OH), 8.17 (dd, J ) 1.5 and 7.9 Hz, 1H), 7.85 (t, J ) 7.7
Hz, 1H), 7.4-7.65 (m, 4H), 7.32 (dd, J ) 3.1 and 9.0 Hz, 1H).
3-Hyd r oxyxa n th on e (5). Meth od A. Compound 5 was
prepared essentially according to Van Allan,17b but with a
simpler workup. A mixture of crude 2,2′,4-trimethoxyben-
zophenone (7) (26.4 g, 97 mmol) and pyridine hydrochloride
(88.2 g, 0.76 mol) was heated (210 °C bath) for 4 h under N2.
The mixture was then poured into ice H2O (200 g), and the
yellow-green precipitate which formed was collected by filtra-
tion, washed with H2O (2 × 100 mL) and 6 N aqueous HCl (3
× 100 mL), and dried in vacuo: yield 16.8 g (81%). Meth od
B. A mixture of 2,2′-dihydroxy-4-methoxybenzophenone (10.0
g, 41 mmol) and pyridine hydrochloride (25.4 g, 0.22 mol) was
heated (210 °C bath) for 6 h to form a melt at gentle reflux
(210-215 °C). The mixture was then poured into ice H2O (80
g), and the NMR-pure precipitate (8.9 g, quantitative) was
recrystallized from hot 95% EtOH: yield 6.3 g (69%); mp 258-
259 °C [lit.17a mp 246 °C]; Rf[benzene-EtOH (20:3)] 0.6; 1H
NMR (CD3SOCD3) δ 11.0 (br s, OH), 8.12 (dd, J ) 1.6 and 7.9
Hz, 1H), 8.02 (d, J ) 8.2 Hz, 1H), 7.77 (td, J ) 1.7 and 7.9 Hz,
1H), 7.63 (d, J ) 8.2 Hz, 1H), 7.46 (td, J ) 1.1 and 7.9 Hz,
1H), 6.92 (dd, J ) 2.1 and 8.2 Hz, 1H), 6.89 (d, J ) 2.1 Hz,
1H).
Eth yl 5-[(9-Oxoxa n th en -2-yl)oxy]va ler a te (8b). Potas-
sium tert-butoxide (4.6 g, 41 mmol) was added in one portion
to a solution of 2-hydroxyxanthone (4) (7.7 g, 36 mmol) in DMF
(50 mL). The resultant suspension (supernatant dark red) was
stirred briefly at 25 °C under N2, and then a solution of ethyl
5-bromovalerate (7.1 mL, 42 mmol) in DMF (20 mL) was added
dropwise over 20 min. The reaction mixture was heated at
for 11 h at 115 °C and then cooled, filtered, and washed with
EtOAc (2 × 10 mL). The filtrate was concentrated to provide
a light-brown oily residue, which solidified after standing
overnight as the neat oil or under hexane. Light-beige crystals
were collected and washed with n-hexane (3 × 10 mL): yield
9.7 g (79%); mp 59-61 °C; Rf[benzene-EtOH (10:9)] 0.79; 1H
NMR (CD3SOCD3) δ 8.22 (dd, J ) 1.6 and 8.0 Hz, 1H), 7.89
(td, J ) 1.6 and 7.5 Hz, 1H), 7.4-7.7 (m, 5H), 4.14 (q, J ) 7.1
Hz, 2H), 4.09 (t, J ) 5.7 Hz, 2H), 2.40 (t, J ) 6.9 Hz, 2H), 1.85
(m, 4H), 1.26 (t, J ) 7.1 Hz, 3H). An analytical sample from
n-hexane-EtOH (10:1) was white needles, mp 58-59 °C.
Anal. Calcd for C20H20O5 (340.36): C, 70.57; H, 5.92.
Found: C, 70.52; H, 5.86.
Eth yl [(9-Oxoxa n th en -3-yl)oxy]a ceta te (9a ). As de-
scribed by Puranik et al.,18b a mixture of 3-hydroxyxanthone
(5) (6.0 g, 28 mmol), ethyl bromoacetate (7.7 mL, 69 mmol),
and anhydrous K2CO3 (25.4 g, 0.18 mol) in dry acetone (500
mL) was refluxed for 12 h and then filtered and concentrated:
yield 7.4 g (88%). Crystallization from EtOH gave the title
product as shiny colorless needles: mp 122-123 °C [lit.15,18a
mp 122-124 °C; lit.18b mp 135-136 °C]; Rf[hexane-EtOH
Anal. Calcd for C13H8O3 (212.20): C, 73.58; H, 3.80.
Found: C, 73.61; H, 3.83.
2,2′,5-Tr im eth oxyben zop h en on e (6). Meth od A.
A
mixture of o-anisoyl chloride (21.8 mL, 0.147 mol) and 1,4-
dimethoxybenzene (37.2 g, 0.27 mol) was heated under N2 for
20 h (200 °C bath). Distillation after HCl evolution subsided
led to recovered 1,4-dimethoxybenzene [18.7 g, bp 92-95 °C
(8 mm), 1H NMR (CDCl3) δ 6.85 (s, 4H), 3.77 (s, 3H)], followed
by the title product at bp 232 °C (7 mm) [lit.17c bp 237-239 °C
(18 mm)], a viscous yellow oil which was suitable for carrying
forward to the next step: yield 25.4 g (63%). Meth od B. A
mixture of o-anisoyl chloride (39 mL, 0.26 mol) and carbon
disulfide (53 mL) was added dropwise over 2 h under ambient
conditions to a stirred mixture of 1,4-dimethoxybenzene (75
g, 0.54 mol, soluble) and anhydrous aluminum trichloride (46
g, 0.35 mol, suspended) in carbon disulfide (95 mL). Reaction
was accompanied by vigorous HCl evolution and a modest
spontaneous exotherm. After 4 h, the reaction was quenched
by pouring the solution into a mixture of ice (1 kg) and 12 N
aqueous HCl (10 mL). The organic layer was washed with
aqueous saturated NaCl (1 L), H2O (2 × 1 L), and again with
aqueous saturated NaCl (1 L), dried (Na2SO4), and distilled
to give recovered 1,4-dimethoxybenzene (40 g), followed by the
title product: yield 61 g (85%); 1H NMR (CDCl3) δ 7.35-7.51
(m, 2H), 6.8-7.1 (m, 5H), 3.78 (s, 3H), 3.68 (s, 3H), 3.57 (s,
3H).
2,2′,4-Tr im et h oxyb en zop h en on e (7). Following Van
Allan,17b a mixture of o-anisoyl chloride (21.8 mL, 0.147 mol)
and 1,3-dimethoxybenzene (35.7 mL, 0.27 mol) was heated
under N2 for 3 h (200 °C bath). Distillation, after HCl
evolution subsided, led to recovered 1,3-dimethoxybenzene [20
g; bp 75-80 °C (5 mm)], followed by title product, bp 241-
243 °C (6 mm) [lit.17b bp 180-200 °C (15 mm); this literature
value is likely an underestimate of the true value], in adequate
purity for carrying forward to the next step: yield 34 g (86%);
Rf[benzene-EtOH (20:3)] 0.9; 1H NMR (CDCl3) δ 7.59 (d, J )
8.6 Hz, 1H), 7.35-7.45 (m, 2H), 6.9-7.1 (m, 2H), 6.45-6.55
(m, 2H), 3.85 (s, 3H), 3.69 (s, 3H), 3.65 (s, 3H).
1
(10:3)] 0.44; H NMR (CDCl3) δ 8.32 (dd, J ) 1.6 and 7.8 Hz,
1H), 8.27 (d, J ) 8.9 Hz, 1H), 7.69 [td, J ) 1.6 and 7.5 Hz,
1H], 7.45 (d, J ) 7.8 Hz, 1H), 7.36 (td, J ) 1.0 and 7.5 Hz,
1H), 6.98 (dd, J ) 2.4 and 8.9 Hz, 1H), 6.87 (d, J ) 2.4 Hz,
1H), 4.74 (s, 2H), 4.30 (q, J ) 7.1 Hz, 2H), 1.31 (t, J ) 7.1 Hz,
3H).
Anal. Calcd for C17H14O5 (298.28): C, 68.45; H, 4.73.
Found: C, 68.65; H, 5.01.
Eth yl 5-[(9-Oxoxa n th en -3-yl)oxy]va ler a te (9b). Anhy-
drous K2CO3 (51.3 g, 372 mmol) was added to a solution of
3-hydroxyxanthone (5) (20.5 g, 97 mmol) plus ethyl 5-bromo-
valerate (53.3 g, 316 mmol) in dry acetone (1.5 L).39 The
reaction mixture was heated to reflux for 8 h, cooled, filtered
to remove inorganic salts, washed with acetone (3 × 50 mL),
and concentrated. The initial brownish crystals were washed
with hexane (3 × 60 mL), providing white crystals which were
dried in vacuo over P2O5: yield 28.5 g (86%); mp 75-77 °C;
1
Rf[hexane-EtOH (10:3)] 0.51; H NMR (CDCl3) δ 8.31 (dd, J
) 1.6 and 7.9 Hz, 1H), 8.23 (d, J ) 8.8 Hz, 1H), 7.67 (td, J )
1.6 and 7.0 Hz, 1H), 7.44 (d, J ) 7.9 Hz, 1H), 7.35 (td, J ) 1.0
and 7.5 Hz, 1H), 6.92 (dd, J ) 2.3 and 8.8 Hz, 1H), 6.84 (d, J
) 2.3 Hz, 1H), 4.06-4.19 (m, 4H), 2.41 (t, J ) 6.8 Hz, 2H),
1.82-1.89 (m, 4H), 1.26 (t, J ) 7.1 Hz, 3H).
Anal. Calcd for C20H20O5 (340.36): C, 70.59; H, 5.92.
Found: C, 70.51; H, 5.83.
[(9-Oxoxa n th en -2-yl)oxy]a cetic Acid , P ota ssiu m Sa lt
a n d F r ee Acid (10a ). Ester 8a (10 g, 33 mmol) was dissolved
in 95% EtOH (150 mL), and 4 N aqueous KOH (30 mL, 0.12
mol) and H2O (15 mL) were added. The mixture was stirred
for 30 min at 35 to 40 °C and then cooled, and the solid
precipitate which formed was collected by filtration, washed
with absolute ether (3 × 20 mL), and air-dried: yield 9.25 g
(96%); Rf[MeOH-H2O (4:1)] 0.90; 1H NMR (CD3SOCD3) δ 8.18
Eth yl [(9-Oxoxa n th en -2-yl)oxy]a ceta te (8a ). A mixture
of 2-hydroxyxanthone (4) (14.1 g, 66 mmol), ethyl bromoacetate
(14.5 mL, 0.13 mol), and anhydrous K2CO3 (53 g, 0.38 mol) in
acetone (350 mL) plus DMF (12 mL) was refluxed for 6 h. The
cooled reaction mixture was filtered to remove inorganic salts,
(39) The text procedure using anhydrous K2CO3 gave much better
yields and purities than when potassium tert-butoxide was used as
base (compare to procedure for 8b).

6336 J . Org. Chem., Vol. 61, No. 18, 1996
Han et al.
(dd, J ) 1.6 and 7.9 Hz, 1H), 7.86 (td, J ) 1.6 and 7.9 Hz,
1H), 7.35-7.65 (m, 5H), 4.19 (s, 2H). The potassium salt
isolated in this way was used directly in the next reaction. A
small portion was converted to the free acid by dissolving in
H2O and acidification with 12 N aqueous HCl to pH ∼1,
providing a quite insoluble light-beige solid, mp 179-182 °C
[lit.18a 180-181 °C]. 1H NMR [DCON(CD3)2] aromatic region
essentially the same (less fine structure) as that described
above for the salt, CH2 shifted to δ 4.94, carboxyl H at δ 13.5
(br).
which appeared after partial concentration of the mother
liquor, and air-dried:40 yield 7.2 g (89%); Rf[EtOH-H2O-
EtOAc (12:1:1)] 0.22 (major spot which became yellow after
spraying with 2% TFA in CH2Cl2); 1H NMR (CD3SOCD3) δ 7.52
(d, J ) 8.0 Hz, 1H), 7.0-7.35 (m, 5H), 6.74 (dd, J ) 3.0 and
8.0 Hz, 1 Hz), 6.2 (s, 1H, exchanges with D2O), 5.61 (s, 1H),
4.12 (s, 2H).
5-[(9-Hyd r oxyxa n th en -2-yl)oxy]va ler ic Acid (12b). A
solution of 1 N aqueous NaOH (5.5 mL, 5.5 mmol) was added
dropwise to dissolve completely keto acid 10b (1.5 g, 4.8 mmol)
in H2O (36 mL); the final pH of the yellow solution was 8.3.
Subsequently, NaBH4 (1.5 g, 39 mmol) was added in a single
portion, and the reaction mixture was stirred for 2.5 h at 50-
55 °C.41a The pale yellow solution was cooled to 10 °C, and
solid NaHCO3 (2.7 g, 10 equiv) was added in small portions
over 30 min.41b After maintenance at 25 °C for 1 h, the solution
was cooled externally to <5 °C, and a mixture of HOAc (10
mL) and ice H2O (10 g) was added [final pH ∼5.5]. A fine
milky white precipitate was collected by filtration, washed with
H2O (3 × 30 mL), and dried in vacuo over P2O5: yield 1.4 g
5-[(9-Oxoxa n th en -2-yl)oxy]va ler ic Acid (10b). A sus-
pension of ethyl ester 8b (6.8 g, 20 mmol) in 95% EtOH (50
mL) was diluted with 4 N aqueous NaOH (50 mL, 0.2 mol).
After 1 h at 25 °C, the reaction mixture was homogeneous;
saponification was quenched after 4 h by the addition with
cooling of 12 N aqueous HCl (17 mL) to adjust the pH to 3. A
light-gray precipitate formed quickly, which was collected,
washed with H2O (3 × 10 mL), and dried in vacuo over P2O5
to give the title product (free acid): yield 6.1 g (94%); mp 151-
1
152 °C; H NMR (CD3SOCD3) δ 8.20 (dd, J ) 1.6 and 8.0 Hz,
1
1H), 7.87 (td, J ) 1.6 and 7.5 Hz, 1H), 7.45-7.7 (m, 5H), 4.09
(t, J ) 5.9 Hz, 2H), 2.31 (t, J ) 7.1 Hz, 2H), 1.6-1.9 (m, 4H).
An analytical sample from n-hexane-EtOH (10:1) was fine
light-beige needles, mp unchanged.
(93%); mp 92-94 °C; Rf[EtOAc-MeOH (2:1)] 0.51; H NMR
(CD3SOCD3) δ 7.61 (d, J ) 6.2 Hz, 1H), 7.32 (t, J ) 6.8 Hz,
1H), 7.0-7.2 (m, 4H), 6.92 (dd, J ) 3.0 and 8.9 Hz, 1H), 5.73
(s, 1H), 4.04 (t, J ) 6.0 Hz, 2H), 2.32 (t, J ) 6.9 Hz, 2H), 1.7-
1.9 (m, 4H); title product41c suitable for direct use in next
reaction.
Anal. Calcd for C18H16O5 (312.31): C, 69.21; H, 5.16.
Found: C, 69.15; H, 5.01.
[(9-Oxoxa n th en -3-yl)oxy]a cetic Acid , F r ee Acid a n d
P ota ssiu m Sa lt (11a ). Ester 9a (3.0 g, 10 mmol) was
dissolved in 95% EtOH (50 mL) at reflux, and 4 N aqueous
KOH (50 mL, 0.2 mol) was added. After 2 h reflux, the alcohol
was evaporated in vacuo, H2O (100 mL) was added, and the
mixture was brought to boiling to dissolve the potassium salt
of the title product. The hot solution was filtered and acidified
with 12 N aqueous HCl while still warm. The title product
(free acid) came out of solution and was collected by filtra-
tion: yield 2.4 g (89%); recrystallized from EtOH to provide
shiny colorless needles, mp 199-201 °C [lit.15,18a,c mp 196-
198 °C; lit.18b mp 204 °C]; Rf[CHCl3-MeOH-HOAc (10:1:0.1)]
0.57; 1H NMR (CD3SOCD3) δ 8.19 (dd, J ) 1.6 and 7.9 Hz,
1H), 8.13 (d, J ) 8.8 Hz, 1H), 7.87 (td, J ) 1.6 and 7.1 Hz,
1H), 7.65 (d, J ) 8.3 Hz, 1H), 7.49 (td, J ) 1.1 and 7.8 Hz,
1H), 7.16 (d, J ) 2.4 Hz, 1H), 7.10 (dd, J ) 2.4 and 8.8 Hz,
1H), 4.94 (s, 2H).
(Xa n th en -3-yloxy)a cetic Acid (13a ). Substrate 11a (0.5
mmol scale) was treated with NaBH4 under the same condi-
tions as for the reduction of the 2-xanthenyl analogue 10a
successfully to the corresponding xanthydrol 12a . Instead of
the expected xanthydrol, the NMR-pure title product was
isolated in 70% yield: mp 172-173 °C; Rf[CH3Cl-MeOH-
1
HOAc (10:1:0.1)] 0.67; H NMR (CD3SOCD3) δ 7.04-7.23 (m,
5H), 6.62 (dd, J ) 2.4 and 8.4 Hz, 1H), 6.55 (d, J ) 2.4 Hz,
1H), 4.48 (S, 2H), 3.96 (s, 2H).
Anal. Calcd for C15H12O4 (256.07): C, 70.29; H, 4.72.
Found: C, 70.11; H, 4.53.
5-(Xa n th en -3-yloxy)va ler ic Acid (13b). Compound 13b
was prepared similarly to compound 13a , but starting from
substrate 11b (0.5 mmol scale) (68% yield): mp 133-135 °C;
Rf[EtOAc-hexane (10:3)] 0.63; 1H NMR (CD3SOCD3) δ 7.03-
7.27 (m, 5H), 6.65-6.68 (m, 2H), 3.97 (m, 4H), 2.29 (t, J ) 6.8
Hz, 2H), 1.69 (m, 4H).
9-F lu or en ylm eth yl Ca r ba m a te (14). Following Stu¨ber
et al.,8c 1.5 N aqueous NH4OH (60 mL, 90 mmol) was added
dropwise with stirring over 10 min to a solution of 9-fluore-
nylmethyl chloroformate (10.3 g, 40 mmol) in 1,2-dimethoxy-
ethane-H2O (4:1, 100 mL). As the ammonia was added, the
title product came out of solution. The reaction endpoint was
indicated by a pH of 7.4, which was maintained for 30 min,
followed by acidification to a pH of 6.5 with 1.0 N HCl (∼1.5
mL). The reaction mixture was chilled in ice, and a white
fluffy solid was collected by filtration, followed by careful
washing with cold H2O (3 × 10 mL) and drying in vacuo over
P2O5: yield 9.4 g (99%); mp 207-208 °C [lit.8c mp 205-206
Anal. Calcd for C15H10O5 (270.23): C, 66.67; H, 3.73.
Found: C, 66.65; H, 3.76.
Alternatively, upon completion of the reflux, more alcohol
(40 mL) was added, and after cooling, the potassium salt of
the title product was collected: yield 2.8 g (91%).
Anal. Calcd for C15H9O5K (308.32): C, 58.43; H, 2.94.
Found: C, 58.45; H, 2.89.
5-[(9-Oxoxa n th en -3-yl)oxy]va ler ic Acid (11b). Ethyl
ester 9b (28.5 g, 84 mmol) was suspended in a mixture of 95%
EtOH (210 mL) and 4 N aqueous NaOH (210 mL); a yellow
homogeneous solution was obtained within 30 min and stirring
continued at 25 °C for a total of 4 h. Partial evaporation to
remove EtOH was followed by addition of H2O (200 mL),
followed by 6 N aqueous HCl under cooling to bring the pH to
3.5. The resultant white precipitate was collected by filtration,
washed with H2O (3 × 50 mL), and dried in vacuo over P2O5:
yield 26.0 g (99%); mp 201-202 °C [lit.18c mp 191-193 °C];
(40) The workup is critical. When this reaction mixture was
quenched with either 10% aqueous HOAc or 10% aqueous citric acid,
over-reduced byproduct (xanthenyl-2-oxy)acetic acid was formed (5%
by HPLC). Characterization after isolation by column chromatogra-
phy: mp 193-194 °C; 1H NMR (CD3SOCD3) δ 7.23 (d, J ) 7.0 Hz,
1H), 7.19 (d, J ) 7.5 Hz. 1H), 6.98-7.05 (m, 3H), 6.81 (d, J ) 2.5 Hz,
1H), 6.78 (dd, J ) 2.5 and 8.5 Hz, 1H), 4.63 (s, 2H), 4.00 (s, 2H). Anal.
Calcd for C15H12O4 (256.07): C, 70.29; H, 4.72. Found: C, 70.19; H,
4.68.
(41) (a) Omission of base was accompanied by uncontrollable foam-
ing, whereas a higher initial pH (i.e., 11-12) significantly retarded
the reaction rate. (b) This step was found to be critical. Quenching
directly into HOAc-ice water was invariably accompanied by the
formation of over-reduced byproduct: 5-(xanthenyl-2-oxy)valeric acid,
mp 149-150 °C; TLC, Rf[EtOAc-MeOH (2:1)] 0.84, [EtOAc-hexane
(10:3)] 0.62; 1H NMR (CD3SOCD3) δ 7.22-7.26 (m, 2H), 6.98-7.08 (m,
3H), 6.78-6.82 (m, 2H), 4.02 (s, 2H), 3.94 (t, J ) 5.7 Hz, 2H), 2.29 (t,
J ) 6.5 Hz, 2H), 1.67 (m, 4H). Anal. Calcd for C18H18O4 (298.12): C,
72.45; H, 6.09. Found: C, 72.28; H, 6.24. An authentic sample of this
over-reduced material was prepared in excellent yield and purity by
quenching the title reaction mixture [which contains xanthydrol 12b]
with 5% aqueous TFA]. (c) The reduced byproduct was absent, as
judged by 1H NMR.
1
Rf[EtOAc-hexane (10:3)] 0.48; H NMR (CD3SOCD3) δ 8.18
(dd, J ) 1.6 and 7.9 Hz, 1H), 8.09 (d, J ) 8.9 Hz, 1H), 7.83
(td, J ) 1.6 and 7.1 Hz, 1H), 7.62 (d, J ) 7.9 Hz, 1H), 7.47 (t,
J ) 7.5 Hz, 1H), 7.15 (d, J ) 2.3 Hz, 1H), 7.04 (dd, J ) 2.3
and 8.8 Hz, 1H), 4.17 (t, J ) 6.0 Hz, 2H), 2.32 (t, J ) 7.0 Hz,
2H), 1.71 (m, 4Η).
Anal. Calcd for C18H16O5 (312.31): C, 69.23; H, 5.16.
Found: C, 69.47; H, 5.26.
[(9-Hyd r oxyxa n th en -2-yl)oxy]a ceta te, Mixtu r e of So-
d iu m a n d P ota ssiu m Sa lts (12a ). A solution of potassium
salt 10a (8.48 g, 27.5 mmol) in H2O (70 mL) was treated with
NaBH4 (2.0 g, 52.8 mmol) which was added in small portions
over 1.5 h while stirring. After 20 h at 25 °C, further NaBH4
(1.0 g, 26.4 mmol) was added, and reduction was continued
for 26 h at 25 °C. The resultant white precipitate was filtered,
washed with EtOH (3 × 50 mL), combined with a second crop

Xanthenylamide Handles for Synthesis of Peptide Amides
J . Org. Chem., Vol. 61, No. 18, 1996 6337
°C]; Rf[benzene-EtOH(10:3)] 0.74; 1H NMR (CD3SOCD3) δ
7.90 (d, J ) 6.8 Hz, 2H), 7.70 (d, J ) 7.0 Hz, 2H), 7.43 (t, J )
7.2 Hz, 2H), 7.34 (t, J ) 7.3 Hz, 2H), 6.6 (br, 2H, NH2), 4.21-
4.29 (m, 3H); 1H NMR (CDCl3) δ 7.76 (d, J ) 6.9 Hz, 2H), 7.60
(d, J ) 7.1, 2H), 7.25-7.40 (m, 4H), 4.71 (br, 2H, NH2), 4.40
(d, J ) 6.9, 2H, CH2), 4.23 (t, J ) 6.9 Hz, 1H, CH).
Ta ble 2. Au tom a ted Dep r otection /Cou p lin g Cycle Usin g
N-[(Dim eth yla m in o)(1H-1,2,3-tr ia zolo[4,5-b]p yr id in -1-yl)-
m eth ylen e]-N-m eth ylm eth a n a m in iu m Hexa flu or o-
p h osp h a te N-Oxid e (HATU)/N,N-Diisop r op yleth yla m in e
(DIEA) or N,N′-Diisop r op ylca r bod iim id e (DIP CDI)/
1-Hyd r oxyben zotr ia zole (HOBt) or
7-Aza -1-h yd r oxyben zotr ia zole (HOAt)a
Anal. Calcd for C15H13NO2 (239.27): C, 75.30; H, 5.48; N,
5.85. Found: C, 75.37; H, 5.60; N, 5.82.
operation
reagent, solvent
min
5-[(9-Am in oxa n th en -2-yl)oxy]va ler ic Acid (15). Start-
ing with keto acid 10b (2.0 g, 6.4 mmol), NaBH4 reduction was
carried out exactly as in the procedure for generating xanthy-
drol 12b. Upon completion of the NaHCO3 quenching step,
(NH4)HCO3 (15.2 g, 30 equiv) was added for a 24 h reaction
at 25 °C. The resultant white suspension was acidified to pH
∼4.5 by dropwise addition of 10% aqueous citric acid, providing
a light-beige product which was collected by filtration, washed
with H2O (3 × 20 mL), and dried in vacuo over P2O5: yield
1.87 g (94%); mp 122-124 °C; Rf[CHCl3-MeOH-HOAc (85:
15:3)] 0.38 (single bright spot under UV); 1H NMR (CD3SOCD3)
δ 7.62 (d, J ) 7.8 Hz, 1H), 7.01-7.31 (m, 5H), 6.85 (dd, J )
2.8 and 8.9 Hz, 1H), 4.94 (s, 1H), 3.97 (t, J ) 5.9 Hz, 2H), 2.26
(t, J ) 6.8 Hz, 2H), 1.7-1.9 (m, 4H).
1
2
3
4a
4b
5
DMF washes
piperidine-DMF (1:4)
DMF washes
1
6
12
30
30
8
AA/HATU/DIEA (1:1:2) in DMFb,c
AA/DIPCDI/HOBt or HOAt (1:1:1) in DMFb,d
DMF washes
a
These operations are performed automatically in the continu-
ous-flow mode on a PerSeptive Biosystems 9050 peptide synthe-
sizer. The indicated reagents were used in 4-fold excess over
b
growing chains on the peptide-PEG-PS supports. c The appropriate
NR-Fmoc-protected amino acid and the HATU reagent were
combined together as solids in the same instrument vial and were
dissolved approximately 6 min before needed to a final concentra-
tion of 0.3 M by automated addition of DIEA (0.6 M) in DMF.
Similar to previous the note, the appropriate solid NR-Fmoc-
protected amino acid and the HOAt or HOBt reagent were
dissolved approximately 6 min before needed to a final concentra-
tion of 0.3 M by addition of DIPCDI (0.3 M) in DMF.
d
Anal. Calcd for C18H19NO4 (313.18): C, 69.01; H, 6.07; N,
4.47. Found: C, 69.28; H, 6.16; N, 4.27.
XAL-r esin s a n d Th eir Use in Solid -P h a se P ep t id e
Syn th esis. Fmoc-XAL (2a ,b and 3a ,b) were introduced
readily onto a variety of amino-functionalized supports. The
parent supports used were either 4-methylbenzhydrylamino-
poly(styrene-co-1% divinylbenzene) (MBHA-PS) resin [Per-
Septive Biosystems (formerly Millipore), initial loading 0.30
or 0.46 mmol/g)] or several polyethylene glycol-polystyrene
(PEG-PS) graft supports (initial loadings 0.10-0.25 mmol/g)
developed in our laboratory and commercially available from
PerSeptive Biosystems; these latter are particularly well suited
for continuous-flow synthesis.22a In general, a norleucine
“internal reference” amino acid residue,6d,42 with its NR-amino
function protected by Boc or Fmoc, was introduced first onto
the support (standard DIPCDI/HOBt in DMF protocol, Table
3), following which the Boc or Fmoc group was removed in the
usual way and the handle was added. In a typical procedure,
handle 3-XAL4 (3b ) (278 mg, 0.52 mmol, 1.3 equiv),
N-[(dimethylamino)(1H-1,2,3-triazolo[4,5-b]pyridin-1-yl)meth-
ylene]-N-methylmethanaminium hexafluorophosphate N-oxide
(HATU) (198 mg, 0.52 mmol, 1.3 equiv), and N,N-diisopropy-
lethylamine (DIEA) (182 µL, 1.04 mmol, 2.6 equiv) were
dissolved in N,N-dimethylformamide (DMF) (3 mL) and added
to PEG-PS support (2.0 g, 0.2 mmol/g, 0.4 mmol, 1.0 equiv)
which had just been subjected to a final wash and filtration
with DMF. The coupling was allowed to proceed in a standard
manual solid-phase reaction vessel by shaking at 25 °C for 16
h, at which point a qualitative ninhydrin test43 indicated that
the reaction had gone to completion. The resin was filtered,
washed with DMF, CH2Cl2, and MeOH (3 × 5 mL × 1 min
per solvent), and then dried in vacuo over P2O5. Fmoc-XAL-
resins were extended further by stepwise automated Fmoc
chemistry with standard HATU/DIEA, DIPCDI/HOBt or HOAt,
or BOP/HOBt/NMM protocols (Tables 2, 3, and 4). Removal
of the Fmoc group from XAL-resins occurred under the same
conditions as Fmoc removal from resin-bound NR-amino groups.
Incorporations of amino acids occurred at the expected integer
ratios. In fact, the “internal reference” amino acid technique
showed that with optimal protocols, nearly quantitative yields
were obtained for all steps, including introduction of the
handle, removal of the handle Fmoc group, and stepwise
incorporation of Fmoc-protected amino acid derivatives. Upon
completion of peptide chain assemblies, aliquots (10-15 mg)
of peptide-resin were treated with a variety of TFA/scavenger/
CH2Cl2 cleavage cocktails (1 mL). Unless specified otherwise,
initial cleavage from the support was carried out for 1 h at 25
°C, following which the filtrate was diluted with an equal
Ta ble 3. Dep r otection /Cou p lin g Cycle Usin g
N,N′-Diisop r op ylca r bod iim id e (DIP CDI)/
1-Hyd r oxyben zotr ia zole (HOBt) or
7-Aza -1-h yd r oxyben zotr ia zole (HOAt)a
operation
reagent, solvent
DMF washes
piperidine-DMF (1:4)
DMF washes
AA/DIPCDI/HOBt or HOAt
(1:1:1) in DMFb,c
time, min
1
2
3
4
1 × 0.3
1 × 1 + 2 × 3
5 × 0.3
30d
5
DMF washes
4 × 0.3
a
b
Protocol for manual operation. The indicated reagents were
used in 4-fold excess over growing chains on the peptide-resin.
c HOBt or HOAt were dissolved in DMF (final concentration ∼0.3
M), and these solutions were used to dissolve the appropriate NR-
Fmoc-protected amino acids. The resultant DMF solutions were
then added to the deprotected peptide-resin, followed by addition
d
of neat DIPCDI. Qualitative ninhydrin tests (ref 43) performed
at this point verified that the coupling reactions had gone to
completion.
volume of an “inverse” cocktail designed to bring the final
concentration of TFA to 50% while maintaining the designated
scavenger concentration. After
a further 1 h at 25 °C,
cleavages were quenched by dilution with HOAc-H2O (4 mL,
3:7), and the CH2Cl2 layers were removed. The aqueous layers
were extracted further with CH2Cl2 (5 × 1 mL) to remove
scavengers, then purged with N2 to remove residual CH2Cl2,
and finally lyophilized to provide crude peptide for HPLC
analysis. Cleavage yields were calculated on the basis of the
amino acid compositions (with respect to Nle “internal refer-
ence”) of the peptide-resins before and after cleavage.
Leu -Ala -Gly-Va l-NH2. Kin etic Stu d ies. Fmoc-XAL (2a ,b
and 3a ,b) were linked to a Phe-PEG-PS support (100 mg per
experiment, 0.21 mmol/g), which comprised PEG:PS in an
approximately 1:1 ratio. Automatic continuous-flow solid-
phase synthesis with Fmoc-amino acids and an HATU/DIEA
protocol (Table 2) provided the appropriate peptide-resins with
satisfactory amino acid analyses for all syntheses (Leu, Ala,
Gly, and Val all 1.00 ( 0.02). The peptide-resins were treated
with TFA-CΗ2Cl2 (1: 19), and samples (∼5 mg) were with-
drawn periodically (abscissa of Figure 1A), washed with
CH2Cl2 (5 × 1 mL), and hydrolyzed in order to determine
cleavage yields (ordinate of Figure 1A). Additionally peptide-
resin derived from 3-XAL4 (3b ) was cleaved with TFA-
CΗ2Cl2 (1: 99) and (1:49), as well as (1:19) (Figure 1B). The
cleaved tetrapeptide amide from these syntheses was of >99%
purity by HPLC: Delta Pak C18 reversed-phase column (3.9
(42) (a) Atherton, E.; Clive, D. L.; Sheppard, R. C. J . Am. Chem.
Soc. 1975, 97, 6584-6585. (b) Matsueda, G. R.; Haber, E. Anal.
Biochem. 1980, 104, 215-227.
(43) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. Anal.
Biochem. 1970, 34, 595-598.

6338 J . Org. Chem., Vol. 61, No. 18, 1996
Han et al.
Ta ble 4. Au tom a ted Dep r otection /Cou p lin g Cycle Usin g
(Ben zotr ia zolyloxy)tr is(d im eth yla m in o)p h osp h on iu m
Hexa flu or op h osp h a te (BOP )/1-Hyd r oxyben zotr ia zole
(HOBt)/N-Meth ylm or p h olin e (NMM) or
1.00; Gly, 1.05. Cleavage with “dilute” reagent R: TFA-
CH2Cl2-thioanisole-1,2-ethanedithiol-anisole (5:85:5:3:2) oc-
curred in the same 90% yield, with similar purity after a
second step to remove side-chain protecting groups.
N,N′-Diisop r op ylca r bod iim id e (DIP CDI)/
Meth od B. A second synthesis started with Fmoc-2-XAL4-
Nle-MBHA-PS-resin (350 mg, 0.44 mmol/g). The experiment
was designed to determine to what extent, if any, the useful
results of part A were due to the nature of the support or to
the loading. The same protection strategy was employed, but
automated synthesis was carried out on a batchwise PerSep-
tive Biosystems 9600 peptide synthesizer with the BOP/HOBt/
NMM protocol (Table 4). The final peptide-resin (502 mg) had
the expected weight gain, and its amino acid composition
matched theory relative to “internal reference” Nle ) 1.08. In
several cleavages, both two stage and single step and with or
without scavengers, the yields (86-92%), purities (>90%), and
amino acid compositions of the desired peptide were indistin-
guishable from the earlier experiment.
CCK-8 Su lfa t e [H -Asp -Tyr (SO3H )-Met -Gly-Tr p -Met -
Asp -P h e-NH2]. Meth od A. Fmoc-3-XAL1 (3a ) was linked to
a PEG-PS support (350 mg, 0.21 mmol/g), which comprised
PEG:PS in an approximately 1:1 ratio. Couplings of Fmoc-
amino acids were performed automatically on a continuous-
flow PerSeptive Biosystems 9050 peptide synthesizer using a
DIPCDI/HOBt coupling protocol (Table 2, operation 4b). The
side-chain carboxyls of aspartate were protected as allyl esters,
and other residues were unprotected. After six cycles of amino
acid addition, the peptide-resin was transferred to a vessel for
manual synthesis, and Fmoc-Tyr(SO3-)-OH‚1/2Ba2+ (210 mg,
5 equiv) was introduced using BOP (147 mg, 5 equiv), HOBt
(105 mg, 10 equiv), and NMM (156 µL, 15 equiv) in DMF (2
mL), 1 h, 25 °C. Additional cycles removed Fmoc, added Fmoc-
Asp(OAl)-OH (DIPCDI/HOBt method, Table 3), and again
removed Fmoc. Upon completion of chain assembly, allyl
esters were cleaved by use of Pd(PPh3)4 (50 mg) in CHCl3-
HOAc-NMM (20:1:0.5) (3 mL), at 25 °C for 2 h. The resin
was next washed (5 mL each) with DMF (3 × 0.5 min),
CH2Cl2 (3 × 0.5 min), DIEA-CH2Cl2 (0.5:99.5) (3 × 2 min),
0.02 M sodium diethyl dithiocarbamate in DMF (3 × 5 min),
DMF (3 × 0.5 min), and CH2Cl2 (3 × 0.5 min), and dried in
vacuo.32 Final cleavage was carried out with TFA-CH2Cl2-
Η2Ã (1:18:1), for 15 min, at 25 °C. The overall cleavage yield
was 71%, and the desired product represented >95% of the
total peptide material, with just traces of the unsulfated
peptide (Figure 7A). Amino acid analysis of the crude peptide
showed the expected ratios: Asp, 2.02; Tyr, 0.95; Met, 1.94;
Gly, 1.05; Phe, 1.04.
Meth od B. Fmoc-2-XAL4 (2b) was linked to a PEG-PS
support (0.15 mg, 0.2 mmol/g), which comprised PEG:PS in
an approximately 1:4 ratio. The side-chain carboxyls of
aspartate were protected as the tert-butyl esters, and other
residues were unprotected. Elongation of the chain was
carried out essentially as described for method A. A variety
of cleavage conditions were examined with the goal to optimize
both the cleavage yield and the relative amount of desired
sulfate peptide. Optimal cleavage (90% yield) was achieved
with TFA-CH2Cl2-â-mercaptoethanol-anisole (50:45:3:2), 15
min, 25 °C. The desired product represented ∼70% of the total
peptide material, with 10% being unsulfated CCK-8 and 20%
being CCK-8 sulfate with one or the other of the aspartates
retaining tert-butyl side-chain protection (Figure 7B). The
amino acid composition of the crude peptide was Asp, 2.11;
Tyr, 1.03; Met, 1.94; Gly, 0.99; Phe, 0.92.
Oxytocin . Chain assembly was carried out batchwise on
a PerSeptive 9600 peptide synthesizer starting with an Fmoc-
2-XAL4-Ala-MBHA-resin (400 mg, 0.08 mmol/g) derived from
2b. The required Fmoc-amino acids (17.0 equiv) were incor-
porated by 1 h couplings (except Ile, 90 min) mediated by
DIPCDI/HOBt (Table 4, operation 4b). Tyrosine was incor-
porated with tert-butyl side-chain protection, whereas aspar-
agine and glutamine were incorporated as the side-chain
unprotected pentafluorophenyl active esters (17.0 equiv for 1
h in the presence of HOBt, no DIPCDI). The final peptide-
resins (0.06 mmol/g) comprised Asp 0.96, Glu 1.01, Pro 1.20,
Gly 0.93, Ile 0.88, Leu 0.98, Tyr 0.89, Cys not determined, Ala
1.05.
1-Hyd r oxyben zotr ia zole (HOBt)a ,b
operation
reagent, solvent
time, min
1
2
3
4a
4b
CH2Cl2-DMF (1:1) washes
piperidine-DMF-toluene (6:7:7)
CH2Cl2-DMF (1:1) washes
10 × 0.3
5 + 8
12
AA/BOP/HOBt/NMM (1:1:1:1) in DMFc 60
AA/DIPCDI/HOBt (1:1:1) in DMFd
60
a
These operations are performed automatically in a batchwise
b
mode on a PerSeptive Biosystems 9600 peptide synthesizer. This
protocol can also be used substituting (7-azabenzotriazolyloxy)-
tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) for
BOP. c The indicated equimolar reagents were used in 8-fold excess
over growing chains on the peptide-resin. The appropriate NR-
Fmoc-protected amino acid, BOP reagent, and HOBt were stored
as solids in the reservoirs of the instrument; they were dissolved
and activated by addition or equimolar NMM (0.2 M) in DMF.
After 2 min at 25 °C, this homogeneous “preactivated” mixture
d
was added directly to the deprotected peptide-resin. The indi-
cated equimolar reagents were used in 17-fold excess over growing
chains on the peptide-resin [this seemingly high excess reflects
the relatively low initial loading for experiments in which this
particular protocol was used]. The appropriate NR-Fmoc-protected
amino acid, DIPCDI reagent, and HOBt were stored as solids in
the reservoirs of the instrument; DMF was added when needed
and after 2 min at 25 °C, a homogeneous “preactivated” mixture
was added directly to the deprotected peptide-resin.
× 150 mm, 5 µm), linear gradient over 20 min of 0.036% TFA
in CH3CN and 0.045% aqueous TFA, from 1:9 to 9:1, flow rate
1.0 mL/min, tR 18.6 min.
F m oc-Ala -P r o-Tr p -Ala -Va l-Le u -Glu (Ot Bu )-Va l-Ala -
NH2. Fmoc-3-XAL4 (3b) was linked to a PEG-PS support (300
mg, 0.21 mmol/g), which comprised PEG:PS in an approxi-
mately 1:1 ratio. Couplings of Fmoc-amino acids were per-
formed automatically on a continuous-flow PerSeptive Bio-
systems 9050 peptide synthesizer using a HATU/DIEA coupling
protocol (Table 2, operation 4a). The side-chain carboxyl of
glutamic acid was protected as the tert-butyl ester, and other
residues were unprotected. The peptide-resin was preswollen
with washes of CH2Cl2 (3 mL, 3 × 5 min), and the cleavage
was performed by alternating washes of TFA-CH2Cl2 (1:99)
(3 mL, 6 × 5 min) and CH2Cl2 (3 mL, 5 × 0.5 min). The
combined filtrates and washes were concentrated by rotary
evaporation. The yield of cleavage was 84%, and the amino
acid composition of the crude cleaved peptide was Glu, 1.01;
Ala, 2.89; Pro, 1.02; Val, 2.04; Leu, 1.04. HPLC analysis
(Figure 2) indicated >90% purity.
T. a tr a tu s Ad ip ok in etic Hor m on e (p Glu -Leu -Th r -P h e-
Th r -P r o-Gly-Tr p -NH2).6b,29 Meth od A. Fmoc-2-XAL4 (2b)
was linked to a PEG-PS support (100 mg, 0.1 mmol/g) prepared
by Dr. J ane L. Chang, which comprised PEG:PS in an
approximately 1:1 ratio. Couplings of Fmoc-amino acids were
performed automatically on a continuous-flow PerSeptive
Biosystems 9050 peptide synthesizer using a DIPCDI/HOBt
coupling protocol (Table 2, operation 4b). The side-chain
hydroxyls of threonine were protected as the tert-butyl ethers,
and the N-terminal pGlu-OH residue was incorporated without
further protection. Amino acid analysis of the completed
peptide-resin showed the expected ratios: Glu, 1.05; Leu, 0.99;
Thr, 1.77; Phe, 0.98; Pro 0.96; Gly, 1.01; Nle, 1.10. Cleavage/
deprotection was carried out in two stages by use of TFA-
CH2Cl2 (1:19), 1 h, 25 °C, followed by filtration and addition
of neat TFA to bring the overall filtrate to TFA-CH2Cl2 (1:1),
1 h, 25 °C. HPLC analysis directly after cleavage (Figure 5A)
showed the presence of desired peptide as well as the two
mono-tert-butyl and the bis-tert-butyl peptide derivatives,
whereas HPLC analysis after full deprotection (Figure 5B)
indicated the desired peptide as the major peak in >94%
purity. The cleavage yield was 89%, and the amino acid
composition of the crude cleaved peptide was Glu, 1.08; Leu,
1.03; Thr, 0.89 (destruction upon hydrolysis); Phe, 1.01; Pro

Xanthenylamide Handles for Synthesis of Peptide Amides
J . Org. Chem., Vol. 61, No. 18, 1996 6339
Oxytocin (reduced form) was obtained by treatment of the
peptide-resin (25 mg) with TFA-CH2Cl2-Et3SiH-H2O (7:93:
0.5:0.5) (2 mL) for 1 h at 25 °C [cleavages were accompanied
by formation of significant levels of cyclized peptide, as
discussed further in the text]. Oxidations were carried out
on peptide-resins (20 mg of peptide-resin) suspended in DMF-
anisole (19:1, ∼0.3 mL) containing Tl(tfa)3 [1.1 to 1.3 equiv,
final concentration ∼0.5 mM]. After 1 to 2 h at 0 °C, the
peptide-resins were washed with DMF and CH2Cl2 (4 × 1 min
each), then cleaved with TFA-CH2Cl2-Et3SiH-H2O
(7:93:0.5:0.5) (2 mL) for 1 h at 25 °C, and worked up in the
standard way. Cleavage yields were typically >90%. Overall
yields of cyclized oxytocin were 35-45%. Analytical HPLC
evaluations showed good purity (e.g., Figure 9). The crude
1987, whose ideas were instrumental in launching the
XAL handle concept. We thank Drs. Roger J . Bontems,
Lajos Gera, and Zhenping Tian for experimental con-
tributions at exploratory stages of this research, Mr.
Balazs Hargittai for useful editorial suggestions, Dr.
J ane L. Chang for supplying PEG-PS used in some of
this work, Drs. Steven Wolff and Arthur M. Felix of
Hoffman-La Roche, Inc. (Nutley, NJ ) for their efforts
in reproducing the preparation and application of
handle 3-XAL4 (3b) on a large scale, Drs. Bruno Kamber
and Bernard Riniker of Ciba-Geigy (Basel, Switzerland)
for helpful discussions and correspondence about the
work in ref 14, and Dr. Antonello Pessi of IRBM (Rome,
Italy) for gracious communications about the patent in
ref 15. Financial support over the years was from the
National Institutes of Health (GM 28934, 42722, and
43552).
peptides were verified against
a standard from Bachem
Biosciences and characterized by FABMS (thioglycerol ma-
trix): calculated monoisotopic mass of C43H66N12O12S2 1006.4,
positive spectrum m/ z 1007.4 [MH+] and 1029.4 [(M + Na)+].
Ack n ow led gm en t. This paper is dedicated to the
memory of Lewis N. Bell, August 29, 1960-May 18,
J O960312D