Organophosphorus and Nitro-Substituted Sulfonate Esters of 1-Hydroxy-7-azabenzotriazole as Highly Efficient Fast-Acting Peptide Coupling Reagents

Article abstract of DOI:10.1021/jo0300183

Organophosphorus esters 9, 10, 14, and 15 prepared via reaction of diethyl- and diphenylphosphoryl chloride, di(o-tolyl)phosphinyl chloride, and 2,8-dimethylphenoxaphosphinyl chloride with HOAt are excellent coupling reagents for peptide synthesis which are generally superior to their uronium/guanidinium analogues and HOBt- or HODhbt-derived phosphate ester counterparts in minimizing loss of configuration during segment coupling. The phosphinyl analogues are more shelf-stable than the phosphoryl systems. The new reagents have been tested in segment couplings leading to two tripeptides (20, 21) and a hexapeptide 22. Outstanding utility is also shown for the solid-phase assembly of the ACP decapeptide. Similar results were obtained with the 2- and 4-nitro- and 2,4-dinitrophenylsulfonyl esters derived from HOAt.

Full text of DOI:10.1021/jo0300183

Or ga n op h osp h or u s a n d Nitr o-Su bstitu ted Su lfon a te Ester s of
1-Hyd r oxy-7-a za ben zotr ia zole a s High ly Efficien t F a st-Actin g
P ep tid e Cou p lin g Rea gen ts†
Louis A. Carpino,* J usong Xia, Chongwu Zhang, and Ayman El-Faham‡
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003
carpino@chem.umass.edu
Received J anuary 13, 2003
Organophosphorus esters 9, 10, 14, and 15 prepared via reaction of diethyl- and diphenylphosphoryl
chloride, di(o-tolyl)phosphinyl chloride, and 2,8-dimethylphenoxaphosphinyl chloride with HOAt
are excellent coupling reagents for peptide synthesis which are generally superior to their uronium/
guanidinium analogues and HOBt- or HODhbt-derived phosphate ester counterparts in minimizing
loss of configuration during segment coupling. The phosphinyl analogues are more shelf-stable
than the phosphoryl systems. The new reagents have been tested in segment couplings leading to
two tripeptides (20, 21) and a hexapeptide 22. Outstanding utility is also shown for the solid-
phase assembly of the ACP decapeptide. Similar results were obtained with the 2- and 4-nitro-
and 2,4-dinitrophenylsulfonyl esters derived from HOAt.
Preventing loss of configuration is one of the major
challenges in peptide synthesis.1 A common method of
minimizing loss of configuration during segment coupling
is to include in the coupling mixture an additive such as
1-hydroxybenzotriazole (HOBt)2 or less often, but usually
more effectively, 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-ben-
zotriazine (HODhbt),3 both described in 1970 by Ko¨nig
and Geiger. The former is also used in many protocols
for stepwise peptide assembly, either in conjunction with
carbodiimides4 or active esters5 or by being incorporated
into stand-alone reagents such as phosphonium6 or
uronium/guanidinium salts.7
Recently, 1-hydroxy-7-azabenzotriazole (HOAt) and its
derived phosphonium and uronium/guanidinium salts8
have been described as more favorable coupling additives
or reagents for both solution-9 and solid-phase synthe-
ses.10 These compounds enhance coupling yields and
reduce the loss of chiral integrity during segment cou-
pling.
While investigating the efficiency of various types of
coupling reagents, a literature survey disclosed several
reports describing the use of a number of organophos-
phorus derivatives of HOBt and HODhbt. These included
diethoxyphosphinyloxybenzotriazole (DepOBt, 1),11 3-(di-
ethoxyphosphinyloxy)-3,4-dihydro-4-oxo-1,2,3-benzotriaz-
ine (DepODhbt, 2),12 3-[O-(2-oxo-1,3,2-dioxaphosphori-
† Abbreviations: ACP ) acyl carrier protein decapeptide 65-74; Aib
) R-aminoisobutyric acid; Bs ) benzenesulfonyl; DB (DMAP) ) 2,6-
di-tert-butyl-4-(N,N-dimethylamino)pyridine; DIEA ) diisopropylethyl-
amine; DmppOAt ) 1-(2,8-dimethylphenoxaphosphinyloxy)-7-azaben-
zotriazole; DNBs ) 2,4-dinitrobenzenesulfonyl; DpopOAt ) 1-(diphe-
noxyphosphoryloxy)-7-azabenzotriazole; DpopODhbt ) 3-(diphenoxy-
phosphinyloxy)-3,4-dihydro-4-oxo-1,2,3-benzotriazene; DpopOBt )1-
(diphenoxyphosphinyloxy)benzotriazole; DpopCl ) diphenoxyphosphoryl
chloride; DppCl ) diphenylphosphinyl chloride; DtpOAt ) 1-[di(o-tolyl)-
phosphinyloxy]-7-azabenzotriazole; DtpOBt ) 1-[di(o-tolyl)phosphinyl-
oxy]benzotriazole; DtpODhbt ) 3-di(o-tolyl)phosphinyloxy]-3,4-dihydro-
4-oxo-1,2,3-benzotriazine; HAPyU ) 1-(1-pyrrolidinyl-1H-1,2,3-triazolo-
[4,5-b]pyridin-1-ylmethylene)-N-methylmethanaminium hexafluoro-
phosphate N-oxide; HATU ) N-[(dimethylamino)-1H-1,2,3-triazolo[4,5-
b]pyridinylmethylene)-N-methylmethanaminium hexafluorophosphate
N-oxide; HBTU ) N-[(1H-benzotriazol-1-yl)(dimethylamino)methyl-
ene]-N-methylmethanaminium hexafluorophosphate N-oxide; HDTU
) O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl)-1,1,3,3-tetramethylu-
ronium hexafluorophosphate; HOAt ) 7-aza-1-hydroxybenzotriazole;
2NBs )2-nitrobenzenesulfonyl; 4NBs ) 4-nitrobenzenesulfonyl; Ns )2-
naphthalenesulfonyl; TCM ) trichloromethane ) chloroform; TIPA )
triisopropylamine; TFE ) trifluoroethanol; TMP ) 2,4,6-collidine
‡ On leave of absence from the Department of Chemistry, Alexandria
University, Alexandria, Egypt.
(4) Rich, D. H.; Singh, J . In The Peptides. Analysis, Synthesis,
Biology; Gross, E., Meienhofer, J ., Eds.; Academic Press: New York,
1979; Vol. 1, p 250.
(5) (a) Ko¨nig, W.; Geiger, R. Chem. Ber. 1973, 106, 3626. (b)
Bodanszky, M. In The Peptides. Analysis, Synthesis, Biology; Gross,
E., Meienhofer, J ., Eds.; Academic Press: New York, 1979; Vol. 1, p
143.
(6) (a) Castro, B.; Dormoy, J . R.; Evin, G.; Selve, C. Tetrahedron
Lett. 1975, 14, 1219. (b) Coste, J .; Le-Nguyen, D.; Castro, B. Tetrahe-
dron Lett. 1990, 31, 205.
(1) For a recent review, see: Lloyd-Williams, P.; Albericio, F.; Giralt,
E. Chemical Approaches to the Synthesis of Peptides and Proteins; CRC
Press: Boca Raton, 1997. See also: (a) Kemp, D. S. In The Peptides.
Analysis, Synthesis, Biology; Gross, E., Meienhofer, J ., Eds.; Academic
Press: New York, 1979; Vol. 1, p 315. (b) Kovacs, J . In The Peptides.
Analysis, Synthesis, Biology; Gross, E., Meienhofer, J ., Eds.; Academic
Press: New York, 1979; Vol. 2, p 485. Benoiton, N. L. In The Peptides.
Analysis, Synthesis, Biology; Gross, E., Meienhofer, J ., Eds.;, Academic
Press: New York, 1983; Vol. 5, p 217.
(7) (a) Dourtoglou, V.; Ziegler, J .-C.; Gross, B. Tetrahedron Lett.
1978, 15, 1269. (b) Dourtoglou, V.; Gross, B.; Lambropoulou, V.;
Zioudrou, C.; Synthesis 1984, 572. (c) Knorr, R.; Trzeciak, A.; Ban-
nwarth, W.; Gillessen, D. Tetrahedron Lett. 1989, 30, 1927.
(8) Carpino, L. A. J . Am. Chem. Soc. 1993, 115, 4397.
(9) For a recent illustration of the use of HATU in rapid peptide
synthesis in solution, see: Carpino, L. A.; Ismail, M.; Truran, G.;
Mansour, E. M. E.; Iguchi, S.; Ionescu, D.; El-Faham, A.; Riemer, C.;
Warrass, R. J . Org. Chem. 1999, 64, 4324.
(2) Ko¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 788.
(3) Ko¨nig, W.; Geiger, R. Chem. Ber. 1970, 103, 2024, 2034.
(10) Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J .
Chem. Soc., Chem. Commun. 1994, 201.
10.1021/jo0300183 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/12/2003
62
J . Org. Chem. 2004, 69, 62-71

Organophosphorus Esters of 1-Hydroxy-7-azabenzotriazole
nanyl)oxy]-3,4-dihydro-4-oxo-1,2,3-benzotriazine (Dop-
ODhbt, 3),12 1,2-benzisoxazol-3-yl diphenyl phosphate 4,13
3-(diphenoxyphosphinyloxy)-3,4-dihydro-4-oxo-1,3-quinazo-
line 5,14 1-diphenoxylphosphinyloxy-2-oxopyridine 6,15
and pentafluorophenyl diphenylphosphinate (FDPP, 7).16
While the present work was underway a report on the
remarkable properties of 2 appeared.17 Although our own
studies on DepODhbt(DEPBT) 2 showed that this re-
agent did not always outperform HATU, we were struck
by how much better DepOBt 1 and DepODhbt 2 per-
formed in terms of configurational control than their
uronium/guanidinium counterparts HBTU and HDTU,
respectively. These findings led us to an examination of
new organophosphorus reagents built upon HOAt, for in
this case the neighboring group effects8 believed to be
important in the properties of HOAt would be superim-
posed on whatever effects enhance the efficiency of the
phosphorus moiety. Based on the results described here,
we believe that these effects are related to the greater
speed with which protected amino acids are converted
to their active esters by the phosphorus derivatives.
reported here freshly prepared material was always used.
Long-term storage, unfortunately, required great care
due to the hydrolytic sensitivity of these materials.
Because these new phosphate esters were clearly superior
to the older uronium/guanidinium reagents for segment
coupling and under certain conditions for solid-phase
peptide assembly as well, it was considered essential to
search for reagents of this type, which exhibited greater
shelf stability.
Having available from other ongoing studies a number
of phosphinyl chlorides such as 1118 and 1219 which were
easy to handle and not particularly moisture-sensitive
relative to the parent model, diphenylphosphinyl chloride
13,20 we investigated the conversion of 11 and 12 to the
corresponding OAt esters. Indeed, as expected the result-
Diethoxyphosphinyloxy-7-azabenzotriazole (DepOAt)
9
and
diphenoxyphosphinyloxy-7-azabenzotriazole
ing esters 14 and 15 proved to be significantly more
stable toward moisture in the air than the corresponding
phosphate esters yet showed the same excellent character
as coupling reagents.
(DpopOAt) 10 were obtained from diethyl phosphorochlo-
ridate or the diphenyl analogue and HOAt in the pres-
ence of triethylamine in dry benzene under a nitrogen
or argon atmosphere at 0 °C (eq 1). Recrystallization of
9 from dry chloroform-hexane and 10 from DCM-hexane
gave analytically pure reagents as colorless blocklike
crystals. The crude products obtained in this way could
be used without further purification, and in the tests
(11) Kim, S.; Chang, A.; Ko, Y. K. Tetrahedron Lett. 1985, 26, 1341.
Previously the abbreviation BDP was used for 1. In the present work,
abbreviations have been used which more easily allow 1, 2, and other
members of the series to be distinguished from one another.
(12) Fan, C.-X.; Hao, X.-L.; Ye, Y.-H. Synth. Commun. 1996, 26,
1455. Previously, the abbreviation DEPBT was used for 2. See the
comment in ref 11 regarding abbreviations.
(13) Ueda, M.; Oikawa, H. J . Org. Chem. 1985, 50, 760.
(14) Horiki, K. In Peptides: Chemistry, Structure and Biology;
Rivier, J . E., Marshall, G. R., Eds.; ESCOM: Leiden, The Netherlands,
1990; p 907.
(15) Kim, S.; Kim, S. S. J . Chem. Soc., Chem. Commun. 1986, 719.
(16) (a) Chen, S.; Xu, J . Tetrahedron Lett. 1991, 32, 6711. (b)
Dudash, J ., J r.; J iang, J .; Mayer, S. C.; J oullie, M. M. Synth. Commun.
1993, 23, 349.
(17) Li, H.; Xiaohui, J .; Ye, Y.; Fan, C.; Romoff, T.; Goodman, M.
Org. Lett. 1999, 1, 91.
Because the greatest stability was found in the Dtp
derivative 14, this system was extended to the corre-
sponding OBt and ODhbt esters 16 and 17, respectively.
The phosphinyl esters proved generally to be as efficient
as the phosphate analogues and in view of their extended
shelf stability are to be recommended for general use.
(18) Harger, M. J . P. J . Chem. Soc, Perkin Trans. 2 1980, 154.
(19) Granoth, I.; Kalir, A.; Pelah, Z.; Bergmann, E. D. Tetrahedron
1970, 26, 813.
(20) Hunt, B. B.; Saunders: B. C. J . Chem. Soc. 1957, 2413.
J . Org. Chem, Vol. 69, No. 1, 2004 63

Carpino et al.
TABLE 1. Ap p r oxim a te Ha lftim es for th e F or m a tion of
Z-Aib-OXt
coupling reagent
t1/2 (DMF) (min)
t1/2 (CDCl3) (min)
DepOAt, 9
DpopOAt, 10
DepODhbt, 2
DpopODhbta
DtpOBt, 16
HATUa
<2
2-3
<2
2-3
7-8
<2
45-47
<2
Active Ester F or m a tion . It is generally believed that
during peptide coupling via uronium/guanidinium or
phosphonium salts, the N-protected carboxylic acid first
reacts with the coupling reagent to form an active ester,
which then reacts with the amino component to give the
corresponding amide. Thus, the speed of formation of
such an active ester is one of the important factors in
evaluating the efficiency of a coupling reagent. The model
chosen for study here involved conversion of N-benzyl-
oxycarbonyl-R-aminoisobutryic acid (Z-Aib-OH, 18) to the
corresponding active ester in both DMF and CDCl3 (eq
2). The benzylic CH2 units of acid 18 (δ 5.09) and active
ester 19 (δ 5.20) were monitored by proton NMR analysis.
Assignment of the peak at δ 5.20 to 19a -c was confirmed
by authentic syntheses of these compounds.21 Because of
the sterically hindered carbonyl group of Aib, activation
in the sense of eq 2 is slow relative to the cases of the
proteinogenic amino acids allowing different coupling
reagents to be more easily differentiated. Results of the
halftime determinations are collected in Table 1. In DMF,
it was found that all of the tested reagents, except for
DtpOBt and DepODhbt, led to rapid formation of the
active esters. Upon switching to the less polar solvent
CDCl3, noticeable reactivity differences were observed,
especially for DtpOBt and HBTU. The case of HDTU
stands out as an exception: the rapid activation caused
by this reagent is not matched by exemplary results in
either segment coupling or peptide assembly.22 Although
not yet completely understood, similar results have been
cited previously and ascribed to the low reactivity of the
active ester.23 These data show that coupling reactions
will be completed much more quickly for the OAt relative
to the ODhbt or OBt systems with the natural high
reactivity of the OAt ester enhanced by its greater ease
of formation.
65-70
<2
11-12 h
14-15
<2
HAPyUa
<2
HDTUa
<2
<2
>24 h
HBTUa
<2
a
See list of abbreviations not defined in text.
TABLE 2. Effect of Cou p lin g Rea gen t on Exten t of
Ep im er iza tion d u r in g [2 + 1] Cou p lin g Lea d in g to
Z-F VP -NH2 a n d Z-GF P -NH2 a n d [3 + 3] Cou p lin g Lea d in g
to Z-GGVAGG-OMe in DMF w ith 2 equ iv of TMP a s
Ba sea ,b
coupling reagent Z-FVP-NH2 Z-GFP-NH2 Z-GGVAGG-OMe
DepOAt, 9
DmppOAt, 15
DtpOAt, 14
HATUc
DepODhbt, 2
DtpODhbt, 17
HDTUc
0.9 (0.9)
3.6 (2.0)
2.9 (1.4)
5.0 (1.8)
3.5
<0.1
0.3
0.4
1.1 (0.9)
0.3
<0.1
2.4
2.4
4.3 (3.6)
8.5 (4.0)
3.3
8.2
DtpOBt, 16
11.4
14.2
2.2
3.6
HBTUc
a
All figures are given as percent of the LDL- or DL-form as
observed by HPLC analysis. Figures in parentheses refer to
b
identical runs but with 1 equiv of the appropriate HOXt added.
c See list of abbreviations not defined in text.
model systems 20-22 were examined. A summary of the
results for the various coupling reagents is given in Table
2. A more extensive listing is presented in the Supporting
Information section (Tables 10 - 12).
It is clear that the new phosphorus-based OAt deriva-
tives are more effective in preserving configuration than
any of the other tested reagents, including HATU. The
best of the previously described uronium/guanidinium
salts (HAPyU)21,24 sometimes equals the results of the
new phosphorus esters, but where differences are ob-
served, the latter have proved more effective in every case
examined to date. Best results were generally, but not
always obtained when 1 equiv of HOXt was included in
the reaction mixture. Since results were best when using
the base 2,4,6-collidine, only results with this base are
included in the summary table. Results for a wide variety
of other bases are found in the Supporting Information.
Among the results gleaned from these tables, similar
to an earlier case24 involving HAPyU, it was found that
for the new reagents DepOAt and DpopOAt a 1 equiv
excess of proline serving as base gave the lowest epimer-
ization levels yet observed for tripeptide 20 in DMF (0.5%
LDL-isomer). Upon switching to other solvents, even
To test for configurational control using the new
phosphorus-based reagents, three previously studied22,24
(21) Carpino, L. A.; El-Faham, A. J . Org. Chem. 1994, 59, 695.
Although the term “active ester” is used in the present work, this term
should be understood to refer to the mixture of species, often including
the corresponding “active amide”, obtained upon activation of the acid.
See: Carpino, L. A.; Imazumi, H.; El-Faham, A.; Ferrer, F. J .; Zhang,
C.; Lee, Y.; Foxman, B. M.; Henklein, P.; Hanay, C.; Mu¨gge, C.;
Wenschuh, H.; Klose, J .; Beyermann, M.; Bienert, M. Angew. Chem.,
Int. Ed. 2002, 41, 441. The latter reference also describes the synthesis
of the O-forms of HATU, HAPyU, and HBTU. In the present work, all
studies of these reagents involved the N-forms.
(22) Carpino, L. A.; El-Faham, A.; Albericio, F. J . Org. Chem. 1995,
60, 3561.
(23) (a) Albericio, F.; Bofill, J . M.; El-Faham, A.; Kates, S. A. J . Org.
Chem. 1998, 63, 9678. (b) See, however, ref 8 and for a more extensive
compilation of pKa data in this series, see: Koppel, I.; Koppel, J .; Leito,
I.; Pihl, V.; Grehn, L.; Ragnarsson, U.; J . Chem. Res., Synop. 1993,
446.
(24) Carpino, L. A.; Ionescu, D.; El-Faham, A. J . Org. Chem. 1996,
61, 2460.
64 J . Org. Chem., Vol. 69, No. 1, 2004

Organophosphorus Esters of 1-Hydroxy-7-azabenzotriazole
TABLE 3. Distr ibu tion of P r od u cts, In clu d in g Va r iou s Deletion P ep tid es, Accor d in g to HP LC An a lysisa for th e
Assem bly of ACP (65-74) via HOAt-Der ived a n d Rela ted Cou p lin g Rea gen ts
equiv of preactivation coupling ACP
entry coupling method reagentsb
time (min)
(min)
(%) -2Ile (%) -Ile72 (%) -Ile69 (%) -Val (%) -Ala (%) -Asn (%)
1
2
3
4
5
6
7
8
9
DepOAt, 9
DpopOAt, 10
DepODhbt, 2
DpopOBtc
HATUc
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
7
7
7
7
7
7
0
0
0
0
0
0
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
84
85
6
2
2
13
26
1
15
4
4
2
1
19
19
1
26
2
1
1
2
3
1
3
4
6
5
3
1
2
10
5
9
21
23
85
38
86
81
< 1d
29
87
30
1
HDTUc
15
DepOAt, 9
DpopOAt, 10
DepODhbt, 2
DpopOBtc
HATUc
2
1
3
7
1
10
11
12
17
15
25
3
19
17
1
22
2
2
3
3
6
4
HDTUc
a
A reversed-phase C-18 column was used with elution by a linear gradient over 20 min of 0.1% TFA in MeCN and 0.1% TFA from 1:19
b
to 1:1, flow rate 1.0 mL/min. Couplings were carried out in DMF in the presence of 2 equiv of DIEA per equivalent of Fmoc-amino
acid/coupling reagent. c See list of abbreviations not defined in text. Only a trace of the desired product was obtained.
d
greater differences were found between the new phos-
phorus reagents and the related uronium/guanidinium
salts. For example, in the special structure-breaking
combination solvent trifluoroethanol/trichloromethane
(TFE/TCM, 1:3)25 12.2% of the LDL-form was obtained for
DpopOAt/TMP as opposed to 38.5% for HATU/TMP. In
DCM in the presence of TMP, 2.2% (DepOAt) and 2.9%
(DpopOAt) were clearly better than values observed for
guanidinium reagents HATU (9.3%) or HAPyU (5.3%).
In the case of a 2:1 mixture of DMF/DCM as solvent in
the presence of TMP, the epimerization figures for
various coupling reagents were found to be less than for
either DMF or DCM alone. For example, in the case of
20, coupling via HATU/TMP in DMF/DCM the amount
of the LDL-form is 2.6% compared to 5.0% in the case of
DMF and 9.3% for DCM.
To check on the coupling efficiency of diphenyl phos-
phorochloridate (DpopCl), various coupling conditions
were used. It was noted that without additive, DpopCl
gave only a very small amount of the desired peptide for
both DIEA and TMP, results which are in sharp contrast
to those of Quibell and co-workers26 on the use of
diphenylphosphinyl chloride (DppCl). If 1 equiv of HOAt
was present, the results were acceptable. Indeed, the
mixture DpopCl/HOAt/Base, which contains DpopOAt as
the active species, gave results which are comparable to
those obtained with the isolated reagent DpopOAt.
Tripeptide 20 was also chosen as a model to study loss
of configuration associated with use of the new organo-
phosphorus reagents under solid-phase conditions. In
comparison with results obtained in solution, the data
show how much more difficult it is to maintain configu-
ration in the solid-phase mode.27 The system involved
overnight coupling of four equivalents of Z-Phe-Val-OH
onto H-Pro-PAL-PEG-PS in the presence of 8 equiv of
TMP in DMF, cleavage of the tripeptide from the resin
via TFA-H2O (9:1) over a period of 1 h, and separation of
the diastereomers as described for the solution system.
Although extensive loss of configuration occurs in all
cases, the data show that the effectiveness of the various
coupling reagents follows the same order as in solution,
thus coupling reagent/LDL (%): DepOAt/11.6, HAPyU/
13.0, HATU/13.6, DpopODhbt/19.1, DepODhbt/19.5, HD-
TU/24.2, HBTU/29.8.
ACP Assem b ly via St ep w ise Cou p lin g on Solid
P h a se. To demonstrate the suitability of the new orga-
nophosphorus-based coupling reagents and compare their
performance with that of the corresponding uronium/
guanidinium analogues in solid-phase syntheses, several
syntheses of the ACP decapeptide segment 65-74 (H-
Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-NH2) were car-
ried out by Fmoc chemistry according to a method
described previously10 in which coupling times are short-
ened and excesses of reagents are reduced in order to
bring out differences among the various reagents studied.
Under these conditions, incomplete incorporations were
detected for Asn onto Gly, Ile72 onto Asn, Ile69 onto Asp,
Val onto Gln, and Ala onto Ala or Asp. The results are
collected in Table 3. Analysis of the chromatograms
indicated that the new HOAt-based organophosphorus
reagents 9 and 10 are as effective as HATU under these
so-called “1.5 × 1.5” conditions with or without preacti-
vation. Under normal coupling conditions such as 4 eqs.
excess amino acid/30 min. coupling time, all reagents
worked well with the exception of HDTU.
Esters of sulfonic acids and HOBt related to those
described for the phosphorus series have also been used
for peptide coupling.28 The reactivity of such sulfonate
esters was shown to be directly related to the presence
of electron-withdrawing substituents in both the HOBt
and the sulfonic acid moieties.29
This methodology has seen little practical application
however, since, depending on the basicity or reactivity
of the amino component of the coupling process, the use
of such sulfonate esters for in situ coupling is often com-
(28) (a) Itoh, M.; Nojima, H.; Notani, J .; Hagiwara, D.; Takai, K.;
Tetrahedron Lett. 1974, 15, 3089; Bull. Chem. Soc. J pn. 1978, 51, 3320,
(b) Devedas, B.; Pandey, R. K.; Mathur, K. B. Ind. J . Chem. 1978, 16,
1026. (c) Kundu, B.; Srivastava, A.; Devadas, B.; Mathur, K. B. Ind.
J . Chem. 1989, 28B, 604. (d) Kundu, B.; Shukla, S.; Shukla, M.
Tetrahedron Lett. 1994, 51, 9613. (e) Khare, S. K.; Singh, G.; Agarwal,
K. C.; Kundu, B. Protein Pept. Lett. 1998, 5, 171.
(29) (a) Furukawa, M.; Hokama, N.; Okawara, T. Synthesis 1983,
42. (b) Topuzzan, N. O.; Matirosyan, M. S. J . Org. Chem. (USSR) 1991,
27, 2148, (c) Devedas, B.; Kundu, B.; Srivastava, A.; Mathur, K. B.
Tetrahedron. Lett. 1993, 34, 6455. (d) Kundu, B.; Agarwal, K. C., J .
Chem. Res., Synop. 1996, 200.
(25) Kurada, H.; Chen, Y.-N.; Kimura, T.; Sakakibara, S. Int. J . Pept.
Protein Res. 1992, 40, 294.
(26) Quibell, M.; Packman, L. C.; J ohnson, T. J . Chem. Soc., Perkin
Trans. 1 1996, 1219.
(27) See also: Carpino, L. A.; El-Faham, A.; Albericio, F. Tetrahe-
dron Lett. 1994, 35, 2279.
J . Org. Chem, Vol. 69, No. 1, 2004 65

Carpino et al.
TABLE 4. Su lfon a te Ester s of HOXta
anal.data: calcd/found
yield
mtd (%)
compd
mp (°C)
1H NMRb
δ
mol formula
C
H
N
Ts-OAt
A
A
A
89.9 133-134
2.51 (s, 3), 7.41-7.49 (m, 3),
7.87-7.90 (m, 2), 8.36-8.41
(dd, 1), 8.78-8.79 (dd, 1)
2.50 (s, 3), 7.38-7.48 (m, 3),
7.55-7.68 (m, 2), 7.76-7.80
(m, 2), 7.99-8.03 (m, 1)
C
C
C
12H10N4O3S 49.65/49.42 3.47/3.31 19.30/19.08
13H11N3O3S 53.97/53.94 3.83/3.66 14.52/14.40
13H12N4O3S 49.65/49.43 3.47/3.25 19.30/19.33
Ts-OBt
69.6 80-81
Ts-4-OAt
Ts-4-Me-OAt
Bs-OAt
72.7 102-103 dec 2.51 (s, 3), 7.39-7.44 (dd, 2),
7.56-7.62 (q, 1), 7.73-7.77
(m, 2), 8.10-8.15 (dd, 1),
8.82-8.84 (dd, 1)
A
A
A
80.5 120-121
88.2 108-109
77.6 84-85
2.51 (s, 3), 2.82 (s, 3),
7.21-7.24 (dd, 1),
C
C
C
12H10N4O3S 51.30/51.23 3.98/3.95 18.45/18.47
11H8N4O3S 47.82/47.54 2.92/2.64 20.28/19.99
12H9N3O3S 52.35/52.20 3.30/3.25 15.26/15.13
7.41-7.45 (d, 2), 7.86-7.90
(d, 2), 8.59-8.61 (d, 1)
7.43-7.49 (q, 1), 7.61-7.69
(m, 2), 7.80-8.04 (m, 3),
8.37-8.42 (dd, 1),
8.75-8.78 (dd, 1)
Bs-OBt
7.41-7.49 (m, 1), 7.55-7.67
(m, 4), 7.78-7.87 (m, 1),
7.90-7.95 (m, 2), 7.99-8.04
(m, 1)
Ms-OAt
A
A
A
A
A
86.2 76-78
70.2 89-90
3.69 (s, 3), 7.48-7.55 (q, 1),
8.43-8.48 (dd, 1), 8.81-8.84
(dd, 1)
3.61 (s, 3), 7.44-7.53 (m, 1),
7.60-7.73 (m, 2), 8.05-8.10
(m, 1)
C6H6N4O3S
C7H7N3O3S
33.64/33.45 2.82/2.56 26.16/25.92
39.43/39.29 3.31/3.17 19.7/19.43
Ms-OBt
2-NBs-OAt
4-NBs-OAt
4-NBs-OBt
88.1 135-136 dec 7.46-7.52 (q, 1), 7.81 (m, 1),
8.00-8.14 (m, 3), 8.39-8.44
C
C
C
11H7N5O5S 41.12/40.97 2.20/2.03 21.80/21.74
(dd, 1), 8.73-8.76 (dd, 1)
78.4 160-161 dec 7.47-7.53 (q, 1), 8.23-8.28
(m, 2), 8.40-8.52 (m, 3),
11H7N5O5S 41.12/41.17 2.20/2.25 21.80/21.81
12H8N4O5S 45.00/45.25 2.52/2.56 17.49/17.56
8.75-8.78 (dd, 1)
72.2 138-139 dec 7.45-7.54 (m, 1), 7.63-7.72
(m, 2), 8.01-8.18 (m, 3),
8.45-8.50 (m, 2)
Ns-OAt
A
A
93.6 140-141
7.5-8.15 (m, 6), 8.5 (dd, 1),
8.65 (dd, 2), 8.74 (dd, 1)
7.2-8.3 (m, 7), 8.65 (d, 2),
8.84 (dd, 1)
C
C
15H10N4O3S 55.21/55.06 3.07/3.12 17.18/17.09
15H10N4O3S 55.21/55.12 3.07/3.06 17.18/17.25
Ns-4-OAt
93.1 135 dec
Ns-ODhbt
2-NBs-4-OAt
A
A
92.1 139 dec
78.4 105 dec
7.40-8.4 (m, 9), 8.65 (d, 2)
7.6-7.7 (m, 1), 7.8-7.92
(m, 1), 8.0-8.2 (m, 4), 8.82
(dd, 1)
7.2-7.6 (m, 2), 8.0-8.2
(m, 2), 8.24-8.70 (m, 2),
8.9 (dd, 1)
C
C
17H11N3O4S 57.78/57.66 3.12/3.06 11.89/11.95
11H7N5O5S 41.12/41.01 2.18/2.10 21.81/21.74
4-NBs-4-OAt
A
B
B
83.5 114 dec
C
C
C
11H7N5O5S 41.12/41.05 2.18/2.18 21.81/21.61
12H8N6O7S 37.90/37.71 2.12/2.13 22.10/21.95
11H6N6O7S 36.07/36.06 1.65/1.58 22.95/23.00
2,4-DNBs-4-Me-OAt
2,4-DNBs-OAt
75.2 156-157 dec 2.83 (s, 3), 7.29 (s, 1),
8.36-8.40 (d, 1), 8.54-8.66
(m, 2), 8.84-8.50 (d, 1)
89.9 147-148 dec 7.51-7.58 (q, 1), 8.30-8.35
(d, 1), 8.48-8.59 (m, 2),
8.64-8.66 (dd, 1),
8.84-8.85 (d, 1)
a
For abbreviations in this table and in Tables 5-9 for the sulfonate moiety see the list of abbreviations not cited in the text. For the
b
method see the Experimental Section. Solvent CDCl3.
promised by formation of a sulfonamide byproduct.28a,29b
Such byproduct formation which, among amino acid
derivatives,30 is particularly prominent with proline
derivatives, can be avoided by preactivation of the
reactive carboxylic acid component.
On the other hand, in the use of these reagents for
segment coupling, especially if such conversions are slow,
use of a preactivation step is counterproductive since loss
of configuration at the C-terminal carboxylic acid residue
directly increases with preactivation time.31 As demon-
strated in other systems, the substitution of 1-hydroxy-
7-azabenzotriazole (HOAt) for HOBt is expected to reduce
the extent of configurational loss, although the advan-
tages of HOAt are lost for long preactivation times. In
the case of application to model tripeptide, 20 which
involves coupling to proline amide, esters 23a or 23b gave
20 in only low yield (ca. 15%), the major product being
sulfonamide 24. For the case involving collidine as base,
(30) For specific use of the methanesulfonate of HOBt as a selective
N-mesylating agent, see: Kim, S. Y.; Sung, N.; Choi, J .; Kim, S. S.
Tetrahedron Lett. 1999, 40, 117.
(31) For the deleterious effect of long preactivation times in other
systems see: (a) ref 27. (b) Han, Y.; Albericio, F.; Barany, G. J . Org.
Chem. 1997, 62, 4307.
66 J . Org. Chem., Vol. 69, No. 1, 2004

Organophosphorus Esters of 1-Hydroxy-7-azabenzotriazole
TABLE 5. Effect of Cou p lin g Rea gen t a n d Ba se on th e Exten t of In ver sion a t Va lin e d u r in g F or m a tion of
Z-P h e-Va l-P r o-NH2 via [2 + 1] Cou p lin g in DMF a
DIEA
LDL, % (yield, %)
TMP
LDL, % (yield, %)
DB(DMAP)
coupling reagent
side peak,b %
side peak,b %
LDL, % (yield, %)
side peak,b %
NsOAt
10.8 (92.1)
37.5 (93.4)
21.2 (91.2)
29.1 (93.1)
19.2 (53.5)
8.1 (59.4)
nd
nd
nd
nd
25.8
10.2
18.9
14.7
0
4.0 (89.9)
33.1 (91.2)
12.6 (89.3)
16.4 (89.9)
5.6 (66.4)
6.2 (68.5)
4.4 (79.3)
4.4 (81.4)
4.7 (81.7)
3.1 (73.4)
12.6 (78.8)
3.9 (71.9)
2.3 (48.5)
NsOAtc
NsOBt
NsODhbtd
2-NBsOAt
2-NBsOAt/HOAt
4-NBsOAt
4-NBsOAt/HOAt
4-NBsOAte
4-NBsOAt/HOAte
4-NBsOBt
8.0
9.1
21.0 (70.4)
7.2 (82.4)
11.1
10.8
7.6
2.5 (80.4)
1.4 (63.7)
13.9
7.5
35.7 (72.5)
12.8 (84.1)
31.0 (70.3)
25.5 (47.7)
14.9 (34.7)
0
7.2
11.4
33.2
37.1
6.1
21.9
35.6
9.6 (85.4)
4.4 (43.1)
1.6 (35.01)
6.8
37.8
38.1
DNBsOAt
DNBsOAt/HOAt
a
Couplings were carried out following published procedures24 with 2 equiv of base being used unless otherwise specified. For HPLC
b
conditions and retention times for the various reaction components see the references cited. The side peak represents the arenesulfonyl
derivative of proline amide formed as a byproduct. In the case of NsOAt, the amide was isolated and characterized (see the Experimental
Section). As noted in previous publications (refs 28a and 29b), such side products are observed only with the more basic amines, e.g.,
â-phenylethylamine and proline; nd ) not determined. c The dipeptide acid was preactivated at 0 °C for 30 min and added to a solution
d
of H-Pro-NH2 and 1 equiv of base in DMF. HPLC analysis in this case showed 20-25% of unreacted starting material. e In these runs,
H-Pro-NH2 (instead of the coupling reagent) was added last to the reaction mixture (preactivated for 3 min).
TABLE 6. Effect of Cou p lin g Rea gen t a n d Ba se on th e
Exten t of In ver sion a t P h en yla la n in e d u r in g F or m a tion
of Z-Gly-P h e-P r o-NH2 in DMF a
Curiously, in the sulfonamidation of proline amide,
there did not appear to be a clear difference between the
reactivity of the sulfonyl esters of HOAt and those of
HOBt as has previously been observed in the case of the
corresponding acyl esters where the OAt esters are
significantly more reactive than the OBt esters.8 Indeed,
results varied depending on the particular system under
study. Thus, whereas the 2-naphthalenesulfonyl and
4-tosyl OBt esters gave the sulfonamides more quickly
than the OAt esters, in the case of the mesyl and
4-nitrobenzenesulfonyl cases the OAt derivatives were
more reactive. Either neighboring group effects are not
important in the sulfonyl systems or other factors take
precedence.32 Indeed sulfonate esters derived from 4-HOAt
were the most reactive of those examined in the present
studies. Currently we have no consistent explanation for
the high reactivity of the 4-OAt sulfonates, since, if
neighboring group effects are not important, it is expected
that the order of reactivity would be RSO2-4-OAt >RSO2-
7-OAt > RSO2OBt in view of the reported pKa data.8,23
While this could explain the high reactivity of the 4-HOAt
derivatives relative to those of 7-HOAt it cannot account
for the inconsistency between the figures for the 7-HOAt
and HOBt derivatives.
coupling reagent
4-NBs-OAt
4-NBs-OAt/HOAt DIEA (2)
4-NBs-OAt TMP (2)
base
yield (%) LDL (%) side peakb (%)
DIEA (2)
30.4
51.6
45.3
48.1
9.9
5.5
2.6
1.1
22.6
9.4
11.3
11.0
4-NBs-OAt/HOAt TMP (2)
a
Coupling conditions and method of HPLC analysis followed
b
the description given in footnote a, Table 5. Arenesulfonyl-Pro-
NH2.
TABLE 7. Effect of Cou p lin g Rea gen t a n d Ba se on th e
Exten t of In ver sion a t r-P h en ylglycin e d u r in g F or m a tion
of Z-P h g-P r o-NH2 in DMF a
coupling reagent
4-NBs-OAt
4-NBs-OAt/HOAt DIEA (2)
4-NBs-OAt TMP (2)
base
yield(%) LDL (%) side peakb (%)
DIEA (2)
61.1
63.5
67.4
73.5
1.2
1.1
3.3
3.0
9.0
9.5
6.9
6.6
4-NBs-OAt/HOAt TMP (2)
a
Coupling conditions and method of HPLC analysis followed
b
the description given in footnote a, Table 5. Arenesulfonyl-Pro-
NH2.
the extent of epimerization was 12.6% for the OBt ester
and 3.9% for the OAt ester. Preactivation for 30 min in
the latter case provided a good yield of 20 but loss of
configuration increased drastically from 3.9 to 33.1%.
Thus, this technique is completely unsuitable for practi-
cal application in any case where sulfonamide formation
is competitive with amide formation.
Of the various sulfonate esters of HOAt, which were
examined (Tables 4-7) only the nitrosubstituted benze-
nesulfonyl esters proved to be useful in reducing epimer-
ization levels equal to or lower than those attained via
the common reagent HATU. For example, whereas
TsOAt/HOAt and BsOAt/HOAt gave in the case of model
tripeptide 20, in the presence of collidine 5.4% and 7.2%
of the LDL-form, respectively, in the case of 4-NBs-OAt/
(32) It is not known whether sulfonate esters are involved in an
equilibrium analogous to that of the O-acyl/N-acyl type which has been
firmly established by X-ray crystallography for the carboxylic acid
derivatives.33 The asymmetric and symmetric stretching vibrations for
the sulfonyl residue measured for MeSO2OAt (1390, 1190 cm-1) or
MeSO2OBt (1387, 1190 cm-1) are more in line with quoted positions34
for sulfonate esters (1420-1330, 1200-1195 cm-1) than for sulfona-
mides (1370-1330, 1180-1160 cm-1). 1-(Methylsulfonyl)benzotriaole
35
shows the corresponding absorptions at 1382 and 1178-1
.
X-ray
crystallographic analysis of MeSO2OBt has confirmed the sulfonate
ester structure.36
J . Org. Chem, Vol. 69, No. 1, 2004 67

Carpino et al.
TABLE 8. Exten t of Rea ction over Tim e for th e
F or m a tion of Active Ester via Rea ction w ith Z-Aib-OH in
th e P r esen ce of DIEAa
TABLE 9. Ra te of F or m a tion of Z-Aib-OXt via Rea ction
of NsOXta
preactivation time (min) NsOAt NsOBt Ns-4-OAt NsODhbt
yield (%) (Z-Aib-OAt or Z-Aib-OBt)
coupling
1
2
39.6
82.2
85.6
94.1
52.6
81.1
90.5
99.5
96.5
100
100
100
3.2
8.6
16.7
37.5
reagent
2 min 5 min 10 min 20 min 30 min 1 h 24 h
5
TsOAt
44.2
56.3
88.1
73.8
79.8
93.3
52.6
66.7
92.9
82.5
86.9
95.1
63.3
78.6
96.4
89.8
93.4
96.0
66.8
82.5
98.1
92.3
95.6
97
100
100
100
81.2 100
89.9 98.9
99.8 100
96.8
30
TsOBt
a
Ts-4-OAtb
BsOAt
77.1
88.0
The rate of formation was followed by reaction of 0.125 mmol
of NsOXt with 0.125 mmol of Z-Aib-OH in 1 mL of DMF. At the
various preactivation times 50 µL was picked up and diluted to 3
mL with a mixture of CH3CN/H2O (1:2) and 10 µL was injected
onto the HPLC column using a linear gradient of 5-65% CH3CN,
in H2O/ 0.1% TFA in 20 min, C18 Nova Pak column (4 µm, 60 Å,
3.9 × 150 mm). The reaction was followed by the rate of decrease
of the starting material (NsOXt) at tR 21.8, 24, 20.8, 23.4 min for
NsOAt, NsOBt, Ns(4OAt), and NsODhbt, respectively, and the rate
of formation of Z-Aib-OXt at tR 19.4, 21.6, 18.8, 21.5 min for Z-Aib-
OAt, Z-Aib-OBt, Z-Aib-4-OAt, and Z-Aib-ODhbt, respectively.
BsOBt
98.8
2-NBsOAt
4-NBsOAt
4-NBsOBt
97.8 100
100 100
100 100
100 100
97 100
97
99.1 100
99.4 99.5
100
99.5 100
100 100
93.6
91.7
100
4-NBs-4-OAtc 97.1 100
MsOAt
MsOBt
86.8
86.2
90.2
88.9
94.9
93.9
a
To a solution of 0.1 mmol of Z-Aib-OH and 0.1 mmol of DIEA
in 1 mL of DMF was added 0.1 mmol of RSO2OAt (or RSO2OBt).
Then 40 µL of the reaction mixture was dissolved in 2.5 mL of
CH3CN and directly analyzed by HPLC. For HPLC separation, a
gradient system was used involving 5-60% CH3CN in H2O/0.1%
TFA in 20 min, flow rate 1 mL/min, λ254 nm, tR: Z-Aib-OAt, 20.1
min; Z-Aib-OBt, 23.1 min; TsOAt, 19.97 min; TsOBt, 22.62 min;
BsOAt, 18.22 min; BsOBt, 21.12 min; 2-NBsOAt, 17.97 min;
4-NBsOAt, 19.38 min; 4-NBsOBt, 21.68 min; MsOAt, 11.09 min;
Con clu sion s
A new class of phosphorus-based coupling reagents
incorporating the HOAt moiety is described which is
superior to the uronium/guanindinium counterparts in
terms of retention of configuration during segment
coupling. Similarly effective sulfonate esters are obtain-
able from nitro substituted sulfonic acids. In contrast to
the uronium/guanindinium counterparts, the new re-
agents are soluble in many organic solvents including a
nonpolar solvent such as DCM.
b
MsOBt, 15.74 min. For HPLC separation, a gradient system was
used involving 5-40% CH3CN in H2O/0.1% TFA in 20 min, flow
rate 1 mL/min, λ254 nm, tR: Ts-4-OAt, 25.83 min; Z-Aib-4-OAt, 26.17
c
min. For HPLC separation, a gradient system was used involving
5-60% CH3CN in H2O/0.1% TFA in 20 min, flow rate 1 mL/min,
λ254 nm, tR: 4-NBs-4-OAt, 18.73 min; Z-Aib-4-OAt, 19.8 min. For
all separations in this table the column was a Waters C18 Novapak,
60 Å, 4 µm column of dimensions 3.9 × 150 mm.
Exp er im en ta l Section
Dieth oxyp h osp h in yloxy-7-a za ben zotr ia zole (Dep OAt,
9). A solution of HOAt (1.36 g, 10 mmol) and TEA (1.39 mL,
10 mmol) in 15 mL of dry benzene was cooled in an ice bath.
To the solution was added dropwise with stirring a solution
of diethyl chlorophosphate (1.72 g, 1.45 mL, 10 mmol) in 10
mL of dry benzene under N2. The addition was completed in
10 min, and stirring was continued at ice-bath temperature
for 1 h and then at room temperature for an additional 4 h.
The reaction mixture was cooled to 5-10 °C, and TEA‚HCl
was removed quickly by filtration. The colorless clear solution
was evaporated to dryness while the temperature was kept
below 35 °C. Dry hexane was added to the oily residue, and
the whole mixture was tightly capped under N2 and placed in
a refrigerator (-20 °C) for 2 h. The oily residue solidified as a
white solid, which was then recrystallized from dry CH2Cl2-
hexane to give 1.74 g (63%) of the ester as colorless plates:
mp 48-50 °C; 1H NMR(CDCl3) δ 8.77 (dd, 1), 8.40 (dd, 1), 7.44
(dd, 1), 4.57 (m, 4), 1.47 (2t, 6); IR (film NaCl) 2987 (m), 1395
(m), 1306 (s), 1026 (vs), 838 (m), 775 (s), 699 (m) cm-1. Anal.
Calcd for C9H13N4O4P: C, 39.71; H, 4.81; N, 20.58. Found: C,
39.84; H, 4.72; N, 20.54.
HOAt the corresponding figure was 3.1% and for DNBs-
OAt/HOAt, 2.3%. If in this system collidine is replaced
by DB(DMAP)24 these figures drop to 1.4% and 1.6%,
respectively. It is to be expected that even lower levels
of configurational loss and lower levels of sulfonamide
byproduct formation will be observed on application of
this methodology to coupling at amino acids other than
proline. For comparison, with HATU in the presence of
1-4 equiv of DB(DMAP) and 1 equiv of HOAt the range
of LDL-isomer formation is 1.5-2.2%.24
Part of the reason for these low epimerization levels
may be the exceptionally rapid rate of activation of the
carboxylic acid component of the reaction induced by
these sulfonate esters. Thus, for conversions of Z-Aib-
OH to Z-Aib-OAt in the presence of DIEA up to 99.1% of
the OAt ester is formed within 2 min with 4-NBs-OAt,
whereas with Ts-OAt at 5 min the comparable figure
is 44.2% and even after 1 h only 81.2% of the acid
has reacted. For selected examples, see Tables 8 and
9.
With such rapid reactions one also expects highly
effective stepwise peptide assembly. In fact, this proved
to be the case for the model decapeptide ACP upon
assembly according to the “1.5 × 1.5” scheme, i.e., with
a 1.5-fold excess of protected amino acid, a 1:1 mixture
of DIEA and collidine as base, and a coupling time of 1.5
min.10 With both 4-NBs-7-OAt and 4-NBs-4-OAt, this led
to syntheses comparable to that achieved by HATU
(Figure 1, Supporting Information). Such excellent re-
sults were not achieved if the mixed base system is
replaced by 2 equiv of DIEA. As noted in earlier studies,
excess DIEA may lead to poorer syntheses due to
increased oxazolone formation and various degradation
reactions including hydrolysis of the active esters.37
D i p h e n o x y p h o s p h i n y l o x y -7 -a z a b e n z o t r i a z o l e
(Dp op OAt, 10). The synthesis was carried out as described
for 9, HOAt (1.36 g, 10 mmol) being reacted with diphenyl
chlorophosphate (Dpop-Cl) (2.68 g, 2.1 mL, 10 mmol) in the
presence of TEA (1.39 mL, 10 mmol) for 2-3 h to give an oily
residue. Dry hexane was added to the oil, and the whole
(33) For example: (a) O-acyl: Vlassi, M.; Germain, G.; Barlos, K.;
Mamos, P.; Refaat, L. S. Z. Kristallagr. 1990, 192, 59. (b) N-acyl:
Barlos, K.; Papaioannou; Voliotis, S.; Prewo, R.; Bieri, J . M. J . Org.
Chem. 1985, 50, 696.
(34) Brown, D. W.; Floyd, A. J .; Sainsbury, M. Organic Spectroscopy;
Wiley: New York, 1988; p 51.
(35) Beveridge, S.; Huppaty, J . L. Aust. J . Chem. 1972, 25, 1341.
(36) Singh, J .; Fox. R.; Wong, M.; Kissick, T. P.; Maniot, J . L.;
Gougoutas, J . Z.; Malley. M. F.; Kocy, O. J . Org. Chem. 1988, 53, 205.
(37) For studies of the stability of HATU in the presence of DIEA,
see: (a) See ref 23a. (b) Gausephol, H.; Behn, C. Peptides 1996. In
Abstracts of the 24th European Peptide Symposium; Ramage, R., Epton,
R., Eds.; Mayflower Scientific, Ltd.: Kingswinford, UK, 1998; p 409.
68 J . Org. Chem., Vol. 69, No. 1, 2004

Organophosphorus Esters of 1-Hydroxy-7-azabenzotriazole
mixture was placed at -20 °C for 2 h. The mixture was
triturated until the oil solidified. Recrystallization of the solid
from dry chloroform-hexane gave 2.32 g (63%) of the ester as
colorless blocklike crystals: mp 62-64 °C; 1H NMR (CDCl3) δ
8.77 (dd, 1), 8.41 (dd, 1), 7.48-7.26 (m, 11); IR (KBr) 3056 (w),
1594 (sh, s), 1492 (s), 1394 (m), 1328 (s), 1184 (tr, s), 1026 (s),
984 (s), 844 (s), 775 (s), 685 (s) cm-1. Anal. Calcd for
C17H13N4O4P: C, 55.43; H, 3.55; N, 15.21. Found: C, 55.20;
H, 3.53; N, 15.28.
was added portionwise to 55 mL of thionyl chloride while
cooling in an ice bath. The mixture was refluxed under a CaCl2
drying tube for 3 h. After removal of excess thionyl chloride
by a water aspirator in a hood, the solid residue was recrystal-
lized from CH2Cl2-hexane to give 15.22 g (97.7%) of the
phosphinic acid chloride as an off-white solid: mp 174-176
°C; 1H NMR (200 MHZ, CDCl3) δ 2.44 (s, 6), 7.18-7.50 (m, 4),
7.87 (d, 2); IR (KBr) 1133 (PdO) cm-1. Previously the chloride
was made by the same method19 but it was not isolated or
characterized. Alternatively, the corresponding phosphine-10-
oxide, mp 161-163 °C, could be directly treated with SOCl2
by exactly the same method to give the chloride in 90.2% yield.
3-(Dip h en oxyp h osp h in yloxy)-3,4-d ih yd r o-4-oxo-1,2,3-
ben zotr ia zin e (Dp op ODh bt). The preparation was carried
out as described for 9, HODhbt (1.63 g, 10 mmol) being reacted
overnight with diphenyl chlorophosphate (2.06 g, 1.73 mL, 12
mmol) in the presence of TEA (1.67 mL, 12 mmol) in 40 mL of
dry CH2Cl2. The resulting pale yellow solution was washed
with cold water (3 × 15 mL) and dried over MgSO4. A pale
yellow oil was obtained after removing the solvent. Dry hexane
was added to the oil and the whole mixture was placed at -20
°C for 2 h. After trituration with a spatula the oil solidified.
Recrystallization from dry chloroform-hexane gave 3.2 g (80%)
2,8-Dim et h ylp h en oxa p h osp h in yloxy-7-a za b en zot r ia -
zole (Dm p p OAt, 15). Meth od A. To a suspension of 0.42 g
(3.054 mmol) of HOAt in 20 mL of anhydrous CH2Cl2 was
added 0.43 mL (1 equiv) of triethylamine with magnetic
stirring. The resulting clear yellow solution was cooled in an
ice bath under an atmosphere of N2 and treated slowly with
0.85 g (1 equiv) of 2,8-dimethylphenoxaphosphinic chloride
(12). The reaction mixture was stirred at 0 °C for 30 min and
then at room temperature for 2 h. After dilution with 30 mL
of CH2Cl2, the organic phase was washed with H2O and
saturated aqueous NaCl (30 mL) and dried over MgSO4. After
removal of solvent with a rotary evaporator with the aid of a
water aspirator, the residue was recrystallized from CH2Cl2-
hexane to give 0.65 g (56.3%) of the phosphinic ester as white
1
of the ester as pale yellow crystals: mp 89-91 °C; H NMR
(CDCl3) δ 8.38 (ddd, 1), 8.21 (ddd, 1), 7.99(m,1), 7.83 (m, 1),
7.44-7.25 (m, 10); IR (KBr) 1718 (vs), 1586 (m), 1488 (s), 1319
(s), 1179 (s), 1017 (m), 945 (s), 836 (s), 746 (s), 677 (m) cm-1
.
Anal. Calcd for C19H14N3O5P: C, 57.72; H, 3.57; N, 10.63.
Found: C, 57.58; H, 3.49; N, 10.58.
1
4,4-Dim eth yld ip h en yl Eth er . p-Cresol (32 mL, 0.3052
mol) and 19.7 g (1.02 equiv) of potassium hydroxide were added
to a 250-mL three-neck flask fitted with a magnetic stirrer,
thermometer, air condenser, and an oil bath. Upon warming
the mixture to 160 °C for 20 min it became dark brown. The
mixture was cooled to 100 °C, and to the solution were slowly
added 1.1 g of very fine copper powder and 36.7 mL (0.2982
mol) of p-bromotoluene, and the mixture was heated at 230-
240 °C for 2 h. After the reaction mixture was cooled to room
temperature, 100 mL of ether was added and the mixture was
filtered using additional ether (60 mL) to wash the inorganic
solid which was precipitated. The combined filtrates were
washed with 2 N aqueous sodium hydroxide (2 × 75 mL),
water, and saturated NaCl (50 mL) and dried over anhydrous
magnesium sulfate. Evaporation of ether by means of a water
aspirator and subsequent fractional distillation (bp 151-156
°C/17 mm) gave di-p-tolyl ether as a colorless oil which quickly
solidified. Recrystallization from MeOH gave 41 g (69.3%) of
the ether as white crystals: mp 50-51 °C (lit.38 mp 50 °C); 1H
NMR (CDCl3) δ 2.31 (s, 6), 6.80 and 7.20 (AA′, BB′ system).
2,8-Dim eth ylp h en oxa p h osp h in ic Acid . By modification
of literature procedures,39 24.8 g of di-p-tolyl ether, 44 mL of
phosphorus trichloride and 21 g of anhydrous aluminum
chloride were placed in a three-neck flask equipped with a
magnetic stirrer and a reflux condenser protected with a CaCl2
drying tube. The mixture was stirred and refluxed overnight.
The reaction mixture was cooled in an ice bath and treated
slowly with 500 g of crushed ice, which caused the precipitation
of a white solid. The solid was removed by filtration and
washed thoroughly with water. The white solid was suspended
in 300 mL of 5% aqueous sodium hydroxide solution in a
beaker containing a magnetic stirrer and treated very slowly
with 20 mL of 30% H2O2. The oxidation reaction was so
exothermic that ice bath cooling was needed. After 15 min the
resulting clear, hot solution was filtered. The filtrate was
cooled in an ice bath and acidified slowly with concentrated
HCl to cause precipitation of 31.62 g (97.2%) of the phosphinic
acid as a white solid: mp >280 °C (lit.39 mp > 300 °C); yield
crystals: mp 164-166 °C dec; H NMR (200 MHz, CDCl3) δ
2.47 (s, 6), 7.22-7.56 (m, 5) 8.09 (d, 2), 8.34 (d, 1), 8.71 (d, 1);
I(KBr) 1128 (PdO) cm-1 31P NMR (300 MHz, CDCl3/85%H3-
;
PO4) δ 23.3 ppm; HREIMS calcd for C19H15N4O3P, M+ 378.0882,
found 378.0877.
Meth od B. To a suspension of 1.25 g of HOAt in 20 mL of
anhydrous CH2Cl2 was added 0.623 g (1 equiv) of imidazole
with magnetic stirring. The resulting white suspension was
cooled in an ice bath under an atmosphere of N2 and treated
slowly with 2.56 g (1 equiv) of 2,8-dimethylphenoxaphosphinic
chloride 12. The reaction mixture was stirred at 0 °C for 30
min, at room temperature for 2 h, and diluted with 30 mL of
CH2Cl2. The reaction mixture was filtered in a sintered glass
funnel over anhydrous MgSO4 under an atmosphere of N2.
After removal of solvent with a rotary evaporator with the aid
of a water aspirator, the residue was recrystallized from CH2-
Cl2-hexane to give 2.86 g (82.3%) of the phosphinic ester as
white crystals, for which the melting point and NMR data
agreed with the data reported above.
Di-o-tolylp h osp h in e Oxid e. Magnesium turnings (13.96
g) were added to 100 mL of anhydrous ether in a three-neck
flask fitted with a condenser, magnetic stirrer, and a dropping
funnel kept under an atmosphere of nitrogen. o-Bromotoluene
(100 g, 0.579 mol) in 100 mL of ether was slowly added to the
mixture. During the addition, the Grignard reaction was
initiated and became so vigorous that ice bath cooling was
needed frequently. After the addition was complete (40 min),
the reaction mixture was refluxed for 15 min and then cooled
with an ice bath and treated slowly with 30.8 mL (0.232 mol)
of diethyl phosphite in 40 mL of ether. The mixture was
refluxed again for 15 min and cooled with an ice bath. Two
hundred and fifty milliliters of 10% HCl and 200 mL of water
were added slowly to the cooled mixture with magnetic
stirring. Ether was evaporated, and the insoluble phosphine
oxide was collected by filtration and recrystallized from CH2-
Cl2-hexane (a few drops of methanol may be added to help
dissolve the solid) to give 39.47 g (73.9%) of the phosphine
oxide as a pale yellow solid: mp 94 °C (lit.40 mp 93-94 °C);
1
73%; H NMR (60 MHz, TFA) δ 2.43 (s, 6), 7.14-7.78 (m, 6);
1
yield 57%; H NMR (60 MHz, CDCl3) δ 2.38 (s, 6), 4.23 (s, 1),
IR (KBr) broad 2600, 2250, 1650 (P-OH), 1145 (PdO) cm-1
.
7.19-7.94 (m, 8); IR (KBr) 2369 (P-H), 1168 (PdO) cm-1
.
2,8-Dim et h ylp h en oxa p h osp h in ic Ch lor id e (Dm p p -
Di-o-tolylp h osp h in ic Acid (Dtp -OH). By modification of
Cl).19 Dimethylphenoxaphosphinic acid (14.53 g 55.84 mmol)
a literature18,40 procedure, to a suspension of 15.04 g of di-o-
(38) Tomita, M. J . J . Pharm. Soc. J pn. 1937, 57, 391; Chem. Abstr.
1939, 33, 21177,8
.
(40) Suggs, J . L.; Freedman, L. D. J . Org. Chem. 1971, 36, 2566.
(41) Petrov, K. A.; Chauzov, V. A.; Mal’Kevich, N. Yu.; Kostrova, S.
M. Zh. Obschch. Khim. 1978, 48, 91.
(39) Freedman, L. D.; Doak, G. O.; Edmisten, J . R. J . Org. Chem.
1961, 26, 284.
J . Org. Chem, Vol. 69, No. 1, 2004 69

Carpino et al.
tolylphosphine oxide in 80 mL of 5 N aqueous NaOH was
treated with 40 mL of 30% H2O2 all at once, and the resulting
mixture was heated on a steam-bath for 20 min. A clear
solution resulted and was filtered while hot. The filtrate was
cooled in an ice bath and acidified slowly with concentrated
HCl, which caused the precipitation of a white solid which was
recrystallized from MeOH-ether to give 13.4 g (83.3%) of the
phosphinic acid: mp 174-176 °C (lit.40 mp 175-177 °C); yield
58-74%; 1H NMR (60 MHz, TFA) δ 2.37 (s, 6), 7.24-8.12 (m,
10 mL), and again DMF (3 × 10 mL). Preactivation was carried
out for 7 min using 25.5 mg (0.04 mmol, 1.5 equiv) of Fmoc-
Asn(Trt)-OH, 15.75 mg (0.04 mmol, 1.5 equiv) of DpopOAt,
and 14.89 µL (0.09 mmol, 3 equiv) of DIEA in 0.3 mL of DMF
in a 4 mL vial. Following the requisite preactivation period (7
min), the solution of the activated amino acid was added to
the resin. The small vial was washed with 0.1 mL of DMF,
and the washing was also added to the above resin. The
resulting resin mixture was allowed to react at room temper-
ature for 1.5 min. The loaded resin was washed with DMF (3
× 10 mL), and the Fmoc group was deblocked with 10 mL of
20% of piperidine in DMF for 7 min. Washing the deblocked
resin with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL), and DMF
(3 × 10 mL) was followed by an analogous coupling step with
Fmoc-Ile-OH. Other amino acids were coupled similarly, and
after the last coupling with Fmoc-Val-OH and deblocking of
the Fmoc group with 20% piperidine in DMF, the loaded resin
was washed with DMF (3 × 10 mL), CH2Cl2 (3 × 10 mL), EtOH
(5 mL), and ether (5 mL). The resin was then treated with 10
mL of 90% aqueous TFA for 2 h, filtered, and washed on the
filter with 10 mL of 10% TFA in CH2Cl2 and 10 mL of CH2Cl2.
The combined filtrates were evaporated to dryness. The crude
product was washed four times with anhydrous ether and
separated by centrifugation. The yield was calculated by the
weight of the crude product. For analysis, 1 mg of the crude
product was dissolved in 1 mL of 0.1% aqueous TFA and
injected directly onto the HPLC column for analysis.
8); IR (KBr) 1143 (PdO) cm-1
.
Di-o-tolylp h osp h in ic Ch lor id e (Dtp Cl, 11).18,41 Di-o-
tolylphosphinic acid (13.5 g) was slowly added to 50 mL of
thionyl chloride with cooling in an ice bath. The mixture was
refluxed under a CaCl2 drying tube for 3 h. After removal of
excess thionyl chloride by a water aspirator in a hood, the oily
residue was fractionally distilled to give 13.55 g (93.4%) of the
phosphinic chloride as a colorless oil (bp 158-165 °C/0.1
mmHg) which solidified quickly (lit.18 bp 150-160 °C/0.08
mmHg): mp 65-66 °C; yield 80.5%; 1H NMR (60 MHz, CDCl3)
δ 2.45 (s, 6), 7.05-8.08 (m, 8); IR (KBr) 1220 (PdO) cm-1
.
Di-o-tolylp h osp h in yloxy-7-a za ben zotr ia zole (Dtp OAt,
14). The preparation was carried out as described for Dmpp-
OAt (method A) to give the ester as a white solid (83.7%): mp
1
170-172 °C; H NMR (CDCl3) δ 2.70 (s, 6), 7.27-7.60 (m, 7),
7.93-8.04 (m, 2), 8.32 (d, 1), 8.74 (d, 1); 31P NMR (300 MHz,
CDCl3/85% H3PO4) δ 46.6; HRIEMS calcd for C19H17N4O2P,
M+ 364.1089, found 364.1106. Anal. Calcd for C19H17N4O2P:
C, 62.64; H, 4.7; N, 15.38. Found: C, 62.23; H, 4.72; N, 15.47.
Di-o-toylph osph in yloxyben zotr iazole (DtpOBt, 16). The
preparation was carried out as described for 14, HOBt (0.1351
g, 1 mmol) being reacted with Dtp-Cl (0.2647 g, 1 mmol) in
the presence of DIEA (0.21 mL, 1.2 mmol) for 5 h in 10 mL of
dry CH2Cl2 to give 0.24 g (66%) of white solid after workup.
Recrystallization from CH2Cl2-hexane gave 0.18 g of an
analytically pure sample of the ester as colorless crystals: mp
Mod el Test Cou p lin g Rea ction s. All test coupling reac-
tions were carried out as previously described with the
phosphorus reagents simply substituted for the uronium/
guanidinium salts: Z-Phe-Val-Pro-NH2,22,24 Z-Phg-Pro-NH2,42
Z-Gly-Phe-Pro-NH2,22 and Z-Gly-Gly-Val-Ala-Gly-Gly-OMe.21,24
Gen er a l Meth od s for th e Syn th esis of Su lfon a te Es-
ter s. Meth od A. To a suspension of HOAt (1 g, 7.35 mmol) or
HOBt (0.99 g, 7.35 mmol) in 30 mL of anhydrous CH2Cl2 was
added 1.04 mL (1 equiv) of triethylamine with magnetic
stirring. The resulting clear yellow solution was cooled in an
ice bath under an atmosphere of N2 and treated slowly with 1
equiv of RSO2Cl. The reaction mixture was stirred at 0 °C for
30 min and then at room temperature for 2 h. After dilution
with 30 mL of CH2Cl2, the organic phase was washed with
water and saturated aqueous NaCl (30 mL) and dried over
MgSO4. After removal of solvent with a rotary evaporator with
the aid of a water aspirator, the residue was recrystallized
from CH2Cl2-hexane to give the sulfonate esters collected in
Table 4.
1
198-200 °C; H NMR (CDCl3) δ 7.84-8.06 (m, 3), 7.50-7.59
(m, 3), 7.30-7.42 (m, 6), 2.62 (s, 6); IR (KBr) 3065 (w), 1593
(s), 1457 (s), 1362 (m), 1278 (m), 1230 (vs), 1151 (s), 1084 (sh,
s), 812 (vs), 774 (sh, vs), 704 (m) cm-1. Anal. Calcd for
C
20H18N3O2P: C, 66.10; H, 4.92; N, 11.56. Found: C, 65.75;
H, 4.97; N, 11.41.
Dtp ODh bt (17) was made according to the method de-
scribed for DmppOAt (method A) from HODhbt and di-o-
tolylphosphinic chloride and obtained as white crystals (76.5%)
after recrystallization from ethyl acetate-ether: mp 178-179
°C dec; 1H NMR (200 MHz, CDCl3) δ 2.59 (s, 6), 7.28-7.39
(m, 4), 7.49-7.57 (m, 2), 7.75-7.83 (m, 1), 7.90-8.15 (m, 4),
8.35 (dd, 1); IR (KBr) 1709 (CdO), 1240 (PdO) cm-1. Anal.
Calcd for C21H18N3O3P: C, 64.45; H, 4.64; N, 10.74. Found:
C, 64.49; H, 4.54; N, 10.69.
Active Ester F or m a tion . To a solution of 0.1 mmol of
Z-Aib-OH 18 and 0.1 mmol of the appropriate coupling reagent
in 0.5 mL of CDCl3 or DMF was added 0.1 mmol of DIEA. The
mixture was immediately transferred to an NMR tube which
was placed in the probe of a Hitachi R-1200 (60 MHz) NMR
instrument. Integration of the 1H NMR peaks at δ 5.1 (acid)
and 5.2 (active ester 19) as the reaction progressed at the probe
temperature (∼37 °C) allowed for rough determination of the
relative rates. The results given in Table 1 are the averages
of at least two runs.
Meth od B. To a suspension of HOAt (1 g, 7.35 mmol) or
HOBt (0.99 g, 7.35 mmol) in 30 mL of anhydrous CH2Cl2 was
added 0.51 g (1 equiv) of imidazole with magnetic stirring. The
resulting pale yellow suspension was cooled in an ice bath
under an atmosphere of N2 and treated slowly with 1 equiv of
RSO2Cl. The reaction mixture was stirred at 0 °C for 30 min
and then at room temperature for 2 h and diluted with 30 mL
of CH2Cl2. The reaction mixture was filtered in a sintered glass
funnel over anhydrous MgSO4 under an atmosphere of N2.
After removal of solvent with a rotary evaporator with the aid
of a water aspirator, the residue was recrystallized from
anhydrous CH2Cl2-hexane to give the sulfonate esters col-
lected in Table 4.
N-2-Na p h th a len esu lfon ylp r olin e Am id e 24. A solution
of Ns-OAt (0.5 mmol) and proline amide (0.5 mmol) in 2 mL
of DMF was stirred at rt overnight. The reaction mixture was
diluted with ethyl acetate (25 mL) and washed with 1 M HCl,
1 M NaHCO3, and satd NaCl (2 × 10 mL each) and dried over
MgSO4. The solvent was removed in vacuo, and the residue
was recrystallized from methylene chloride and hexane (1:4)
as white crystals in 89.1% yield: mp 130-131 °C; IR (KBr)
3446 (NH), 1675 (CO, amide) 1337, 1184 (SO) cm-1; 1H NMR
(CDCl3) δ 1.8-2.2 (m, 4), 3.2 (t,2), 4.3 (m, 1), 5.6-5.8 (m, 2)
7.4-8.2 (m, 5) 8.5 (d, 2). Anal. Calcd for C15H16NO3S: C, 59.21;
H, 5.26; N, 9.20. Found: C, 58.96; H, 5.34; N, 9.12.
Assem b ly of ACP (65-74) via St ep w ise Solid -P h a se
Cou p lin g.10 Run 2 of Table 3 is taken as an example to
demonstrate the standard protocol used: 150 mg of Fmoc-Gly-
PAL-PEG-PS resin (0.19 mmol/g, 0.0285 mmol) in a 10 mL
disposable syringe fitted with a Teflon filter was washed with
CH2Cl2 (3 × 10 mL) and DMF (3 × 10 mL) and deprotected
with 20% piperidine in DMF (10 mL) for 7 min. The depro-
tected resin was washed with DMF (3 × 10 mL), CH2Cl2 (3 ×
(42) Wenschuh, H.; Beyermann, M.; Haber, H.; Seydel, J . K.; Krause,
E.; Bienert, M. Carpino, L. A.; El-Faham, A.; Albericio, F. J . Org. Chem.
1995, 60, 405.
70 J . Org. Chem., Vol. 69, No. 1, 2004

Organophosphorus Esters of 1-Hydroxy-7-azabenzotriazole
Ack n ow led gm en t. This work was supported by the
National Institutes of Health (GM-09706) and the
National Science Foundation (CHE-0078971). The Na-
tional Science Foundation is also thanked for grants
used to purchase the high-field NMR spectrometers
used in this research.
suggests that mixtures of O- and N-phosphorylated
derivatives were formed. In our work, we assumed the
formation of O-substituted species. For the previously
described compound 2 the O-structure has been con-
firmed by X-ray crystallography (ref 17).
Su p p or tin g In for m a tion Ava ila ble: Figure 1 and tables
detailing results on segment coupling reactions, appropriate
spectral data for new compounds, and HPLC curves for various
samples of the synthetic ACP decapeptide. This material is
available free of charge via the Internet at http://pubs.acs.org.
Not e Ad d ed a ft er P r oof. After the submission of
this paper, we located a reference (Hoffmann, F.; J a¨ger,
L.; Griehl, C. Phosphorus, Sulfur Silicon 2003, 178, 299)
which describes the synthesis of a number of phosphorus
derivatives of N-hydroxy compounds, including our com-
pound 10. These authors cite 31P NMR data which
J O0300183
J . Org. Chem, Vol. 69, No. 1, 2004 71