Boro-norleucine as a P1 residue for the design of selective and potent DPP7 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2005.06.076

Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) α-amino acids in the P2 position and boro-norleucine (boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a DPP7 IC₅₀ value of 480 pM.

Full text of DOI:10.1016/j.bmcl.2005.06.076

Bioorganic & Medicinal Chemistry Letters 15 (2005) 4256–4260
Boro-norleucine as a P1 residue for the design of selective
and potent DPP7 inhibitors
Kevin R. Shreder,* Melissa S. Wong, Sergio Corral, Zhizhou Yu, David T. Winn, Min Wu,
Yi Hu, Tyzoon Nomanbhoy, Senaiet Alemayehu, Stacy R. Fuller,
Jonathan S. Rosenblum and John W. Kozarich
ActivX Biosciences, 11025 N. Torrey Pines Road, La Jolla, CA 92037, USA
Received 20 April 2005; revised 20 June 2005; accepted 23 June 2005
Available online 9 August 2005
Abstract—Dipeptide-based inhibitors with C-substituted (alkyl or aminoalkyl) a-amino acids in the P2 position and boro-norleucine
(boro-Nle) in the P1 position were synthesized. Relative to boro-proline, boro-Nle as a P1 residue was shown able to significantly
dial out DPP4, FAP, DPP8, and DPP9 activity. Dab-boro-Nle (4g) proved to be the most selective and potent DPP7 inhibitor with a
DPP7 IC50 value of 480 pM.
Ó 2005 Elsevier Ltd. All rights reserved.
Proline-specific serine hydrolases have become a widely
studied enzyme class due to growing evidence that they
play an important role in various metabolic functions.
The dipeptidyl peptidases (DPPs) are a subclass of the
serine protease family that cleaves dipeptides from the
amino terminus of proteins and prefers a proline residue
in the penultimate position. Members of the DPP family
include DPP4, DPP7, fibroblast activation protein a
(FAP), DPP8, and DPP9.1
Pro. In contrast, Pro and Ala, in that order, were found
to be preferred P1 residues for DPP4. In addition, the
DPP4 S2 site was found tolerant of a wide variety of
P2 residues. This study indicated that there was a dis-
tinction between the S1 and the S2 subsites of these
DPPs which, in turn, could be exploited to synthesize
selective DPP7 inhibitors.
Inhibitors of DPP7 have been discovered as the result of
both indirect and targeted efforts. As part of counter-
screening efforts during the design of DPP4 inhibitors,
some researchers have identified sub-lM DPP7 inhibi-
tors based on boro-proline (boro-Pro) dipeptides,5
substituted cycloalkylglycines,6 homophenylalanine,7
substituted piperazines,8 and fluoropyrrolidine amides.9
DPP7 active inhibitors based on thioxo amides of pyrro-
lidides and thiazolidides,10 phosphonates,11 and boro-
Pro12 have also been discovered while profiling these
compounds against multiple prolylpeptidase family
members. Most deliberately, Augustyns and co-workers
designed DPP7 inhibitors taking advantage of the P2
preference of this enzyme for cationic sidechains. (S)-
2,4-diaminobutanoic acid (Dab) was found to be a po-
tent and selective DPP7 active P2 residue. For example,
1-[Dab]-piperidine was shown to have a DPP7 IC50 val-
ue of 130 nM and a more than 7500-fold selectivity over
DPP4.13,14 Further optimization of this P2 residue has
yielded c-amino-arylalkyl-substituted Dab analogs with
sub-nM potency for DPP7 and a more than million-fold
selectivity versus DPP4.15
DPP7 is a 58 kDa glycoprotein with an enzymatic func-
tion similar to DPP4 but only distantly related by
sequence homology.2 Huber and co-workers have
reported that inhibition of DPP7 activity induces apop-
tosis in quiescent lymphocytes.3 As part of an effort to
identify the substrate specificity of DPP7, Leiting,
et al. synthesized a positional scanning library of fluo-
rescent dipeptide substrates to identify preferred P1
and P2 residues.4 Not surprisingly, it was found that
DPP7 has an almost absolute specificity for proline in
the P1 position. The second most preferred P1 residue
for DPP7 was determined to be norleucine (Nle).
Furthermore, DPP7 was shown to have a preference in
order at the P2 position for Lys, Nle, Met, Ala, and
Keywords: Dipeptidyl peptidase; Boro-norleucine; DPP4; FAP; DPP7;
DPP8; DPP9.
*
Corresponding author. Tel.: +1 8585262517; fax: +1 8585874878;
e-mail: kevins@activx.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.076

K. R. Shreder et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4256–4260
4257
Herein, we report the synthesis and DPP inhibitory
activity (DPP4, DPP7, FAP, DPP8, and DPP9) of a
novel series of dipeptide-based inhibitors with C-substi-
tuted a-amino acids in the P2 position and boro-norleu-
cine (boro-Nle) in the P1 position.
Val-boro-Nle (4e) could be compared to its straight chain
congener 4c. In both the cases, the inhibitory activity
against DPP7 was similar, but there was a 21-fold reduc-
tion in DPP4 potency for the former compound. When
the cyclic P2 containing Pro-boro-Nle (5) was examined,
activity against DPP7 was diminished 10-fold compared
to its Ôring-openedÕ congener 4c. For Pro-boro-Nle (5),
no inhibition of DPP4 was observed.
Boro-Nle inhibitors were prepared as shown in Scheme 1
using methodology developed by Matteson for the
synthesis of chiral amino boronic acids.16 The commer-
cially available butyl boronic acid was converted to the
boronic ester 1 by stirring with (+)-pinanediol in diethyl
ether. Homologation with (dichloromethyl)lithium and
catalytic zinc chloride yielded the a-chloro boronic
pinanediol ester 2 in 55% yield.17 Nucleophilic displace-
ment of the chlorine atom by lithium hexamethyldisilaz-
ane gave the bis(trimethylsilyl)-protected amine 3 that
was purified using silica gel chromatography (0–10%
EtOAc/hexanes) with an isolated yield of 27%. Treat-
ment of compound 3 with dry HCl saturated hexanes
at 0 °C afforded the hydrochloride salt, which was taken
on without further purification. The reaction of this salt
with various Boc-protected amino acids under standard
peptide coupling conditions generated the corresponding
Boc/pinanediol protected inhibitors. After removal of the
Boc group with 4 N HCl/ dioxane, the pinanediol ester
was deprotected through transesterification with phen-
ylboronic acid using a biphasic hexane/water system.18
The aqueous layer containing the free boronic acid was
loaded onto a Dowex 50-WX2-100 ion-exchange col-
umn, washed with water, and the product was eluted with
2% ammonium hydroxide. The resulting free base form
was converted to the HCl salt using 4 N HCl/dioxane.
When compared to their straight chain alkyl analogs, P2
aminoalkyl derived boro-Nle inhibitors (4f–4i) were
more potent inhibitors of DPP7. Compounds 4f, 4h,
and 4i were found to have similar single digit nM poten-
cies against DPP7 with IC50 values of 8.2, 4.7, and
3.7 nM, respectively. Of all the boro-Nle-based inhibi-
tors, the aminoethyl P2 containing Dab-boro-Nle (4g)
was the most potent against DPP7 with an IC50 value
of 480 pM. Interestingly, the DPP7/4 selectivity was
found to be >68,000, making Dab-boro-Nle (4g) the
most selective DPP7 inhibitor between these two DPPs.
The FAP, DPP8, and DPP9 inhibitory activities for all
of the boro-Nle inhibitors were also examined. No sig-
nificant inhibition of FAP activity was observed for
any of the inhibitors. For DPP8, the alkyl series yielded
two inhibitors (4b and 4e) with sub-lM DPP8 potencies
of 400 and 190 nM, respectively. Both compounds are
structurally related (S)-ethylglycine derivatives. With a
DPP8 IC50 value of 350 nM, Lys-boro-Nle (4i) was iden-
tified as the most potent DPP8 inhibitor in the amin-
oalkyl series. For DPP9, potency increased as length
increased in both the straight chain alkyl and the amin-
oalkyl series. Among the inhibitors with quantifiable
values, Dab-boro-Nle (4g) was the most selective
DPP7 inhibitor when contrasted against DPP8 and
DPP9. For this inhibitor, DPP7/8 and DPP7/9 selectivi-
ty factors of 9000 and 1700 were noted, respectively. In
summary for the latter of the three DPPs, no P2 residue
was found to deliver a compound that inhibited FAP
activity. In the case of DPP8 and DPP9, the choice of
P2 residue played a critical role in determining potency.
Two classes of boro-Nle-based inhibitors were synthe-
sized; those with alkyl (4a–4e and 5) and aminoalkyl
(4f–4i) C-substituted a-amino acids in the P2 position
(see Table 1). IC50 values for these compounds were deter-
mined against five members of the DPP family: DPP7,
DPP4, FAP, DPP8, and DPP9.19 Among the straight
chain alkyl variants (4a–4d), Nle-boro-Nle (4d) was the
most potent inhibitor for both DPP7 and DPP4 with
IC50 values of 18 and 370 nM, respectively. The activities
of the two other types of alkyl P2 residues, Val and
Pro, were also examined. The P2 b-alkyl branched
To determine the impact on selectivity of boro-Nle as a
P1 residue versus boro-Pro, boro-Pro congeners of
HO
HO
LiHMDS
LiCHCl2
O
OH
O
Cl
B
B
B
THF, -78 oC to r.t., 18 h
27%
THF, -78 oC to r.t., 18 h
55%
Et2O, r.t., 3h
98%
O
OH
O
1
2
1) 4N HCl/dioxane
2) phenylboronic acid,
hexanes/water
O
O
1) HCl (g)/hexanes
Si
BocHN
H2N
O
O
OH
N
B
N
B
N
B
H
H
2) Boc-AA-OH,
EDC, HOBt, NMM
DCM, r.t., 16 h
3) Dowex 50WX2-100
4) 4N HCl/dioxane
Si
O
R
O
R
OH
3
Scheme 1. Synthesis of boro-Nle inhibitors.

4258
K. R. Shreder et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4256–4260
Table 1. DPP4, FAP, and DPP7 inhibition data for compounds 4a–i and 5
O
O
H
N
H2N
OH
OH
N
H
B
N
H
B
R
OH
OH
4a-i
5
Compound
R
IC50s (nM)a
FAP
DPP7
DPP4
DPP8
DPP9
4a
4b
4c
4d
4e
4f
4g
4h
4i
CH3
CH2CH3
220
49
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
ni
6000
400
5200
1000
190
ni
40% (20 lM)b
220
32,000
810
370
CH2CH2CH3
CH2CH2CH2CH3
CH(CH3)2
71
18
160
65
40
17,000
2000
40% (33 lM)b
1200
510
4300
CH2NH2
8.2
0.48
4.7
3.7
890
CH2CH2NH2
CH2CH2CH2NH2
CH2CH2CH2CH2NH2
—
4300
2600
350
ni
800
400
ni
280
35% (20 lM)b
5
ni
a ni = no inhibition (i.e., no significant inhibition observed at concentrations as high as 33 lM (DPP7, DPP4, and FAP) or 20 lM (DPP8 and DPP9)).
b Percent inhibition at the concentration indicated.
inhibitors 4d and 4g (Table 2) were synthesized from the
(+)-pinanediol ester of (R)-boro-Pro and the Boc pro-
tected versions of Nle or Dab according to literature
procedure.5 The resulting Nle-boro-Pro (6a) and Dab-
boro-Pro (6b) were potent inhibitors of DPP7 with
IC50 values of 2.0 and 0.72 nM, respectively.20 Both
compounds were also potent inhibitors of DPP4, which
resulted in only modest DPP7/4 selectivity factors (2.2
and 21, respectively). By comparison, Nle-boro-Nle
(4d) and Dab-boro-Nle (4g) yielded DPP7/DPP4 selec-
tivity factors of 21 and >68,000. Similar trends were seen
for FAP, DPP8, and DPP9; derivatization with boro-
Pro resulted in significantly more potent inhibitors for
these three DPPs versus derivatization with boro-Nle.
In summary, the use of boro-Nle over boro-Pro as a
P1 residue can greatly increase DPP7 selectivity.
chose to examine the role that the boronic acid played
in DPP potency. The n-pentyl amides of Nle (7a) and
Dab (7b) were synthesized from n-pentylamine and the
Boc protected versions of these amino acids using the
coupling and Boc deprotection conditions outlined in
Scheme 1. These compounds were designed as controls
lacking the boronic acid moiety for direct comparison
to Nle-boro-Nle (4d) and Dab-boro-Nle (4g). The
DPP7 IC50 values for the compounds 7a and 7b were
5500 and 2600 nM, respectively. These values represent
a 300- and 5400-fold reduction in DPP7 potency relative
to their boronic acid containing congeners 4d and 4g. In
addition, compounds 7a and 7b did not significantly
inhibit the activity of any other DPP. Thus, the presence
of the boronic acid contributes significantly to the DPP
potency of inhibitors 4d and 4g.
Since potent DPP7 inhibitors without an electrophilic
functionality were known (e.g., 1-[Dab]-piperidine), we
Because alanine has been previously identified as an
acceptable P1 residue for DPP4 substrates,21 we specu-
Table 2. DPP4, FAP, and DPP7 inhibition data for compounds 6a through 8.
O
OH
N
B
H2N
OH
O
N
B
H2N
OH
H2N
H
O
OH
N
H
R
R
6a-c
7a-b
8
Compound
R
IC50s (nM)a
FAP
DPP7
DPP4
DPP8
DPP9
6a
6b
6c
7a
7b
8
CH2CH2CH2CH3
CH2CH2NH2
CH(CH3)2
2.0
0.72
4.5
15
4100
30
95
4.6
6.7
2
6800
11
38
0.30
35% (33 lM)b
ni
0.89
15
CH2CH2CH2CH3
CH2CH2NH2
—
5500
2600
160
ni
ni
ni
ni
22,000
ni
670
ni
3200
a ni = no inhibition (i.e., no significant inhibition observed at concentrations as high as 33 lM (DPP7, DPP4, and FAP) or 20 lM (DPP8 and DPP9)).
b Percent inhibition at the concentration indicated.

K. R. Shreder et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4256–4260
4259
Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem. Lett.
2004, 14, 43; (b) Ashton, W. T.; Dong, H.; Sisco, R. M.;
Doss, G. A.; Leiting, B.; Patel, R. A.; Wu, J. K.; Marsilio,
F.; Thornberry, N. A.; Weber, A. E. Bioorg. Med. Chem.
Lett. 2004, 14, 859.
lated that a shortening of the alkyl chain length of boro-
Nle to the corresponding boro-alanine (boro-Ala) would
improve DPP4 potency. Val-boro-Ala (8) was synthe-
sized from methylboronic acid in an analogous fashion
to the boro-Nle synthesis described earlier. In agreement
with the proposed influence of the P1 chain length, Val-
boro-Ala (8) is a potent inhibitor of DPP4 with a DPP4
IC50 value of 0.89 nM. In contrast, the potency of Val-
boro-Nle (4e) against DPP4 was some 19,000-fold less.
For both the compounds 4e and 8, the DPP7 potency
differed by a factor of 4 with DPP7 IC50 values of 40
and 160 nM, respectively.22 Thus, a shorter P1 chain
length can significantly increase DPP4 potency but
may have a less profound effect on DPP7 potency.
7. Edmondson, S. D.; Mastracchio, A.; Beconi, M.; Colwell,
L. F., Jr.; Habulihaz, B.; He, H.; Kumar, S.; Leiting, B.;
Lyons, K. A.; Mao, A.; Marsilio, F.; Patel, R. A.; Wu, J.
K.; Zhu, L.; Thornberry, N. A.; Weber, A. E.; Parmee, E.
R. Bioorg. Med. Chem. Lett. 2004, 14, 5151.
8. Brockunier, L. L.; He, J.; Colwell, L. F., Jr.; Habulihaz,
B.; He, H.; Leiting, B.; Lyons, K. A.; Marsilio, F.; Patel,
R. A.; Teffera, Y.; Wu, J. K.; Thornberry, N. A.; Weber,
A. E.; Parmee, E. R. Bioorg. Med. Chem. Lett. 2004, 14,
4763.
9. Caldwell, C. G.; Chen, P.; He, J.; Parmee, E. R.; Leiting,
B.; Marsilio, F.; Patel, R. A.; Wu, J. K.; Eiermann, G. J.;
Petrov, A.; He, H.; Lyons, K. A.; Thornberry, N. A.;
Weber, A. E. Bioorg. Med. Chem. Lett. 2004, 14, 1265.
10. Sto¨ckel-Maschek, A.; Mrestani-Klaus, C.; Stiebitz, B.;
Demuth, H.-U.; Neubert, K. Biochem. Biophys. Acta 2000,
15, 1479.
The selectivity profiles of both these compounds were
also compared to Val-boro-Pro (6c). For DPP7, IC50
values for all three compounds did not differ by a factor
of more than 4. For the other DPPs, when compared to
compounds 4e and 8, Val-boro-Pro (6c) was found to be
the most potent inhibitor and, in some cases, by several
orders of magnitude. Using the P2 residue Val as an
example, it is clear that DPP selectivity and potency
can sometimes best be modulated by choosing amino
boronic acids other than boro-Pro for the P1 position.
11. Senten, K.; Danie¨ls, L.; Van der Veken, P.; De Meester, I.;
´
Lambeir, A.-M.; Scharpe, S.; Haemers, A.; Augustyns, K.
J. Comb. Chem. 2003, 5, 336.
12. Hu, Y.; Ma, L.; Wu, M.; Wong, M. S.; Li, B.; Corral, S.;
Yu, Z.; Nomanbhoy, T.; Alemayehu, S.; Fuller, S. F.;
Rosenblum, J. S.; Rozenkrants, N.; Minimo, L.; Ripka,
W. C.; Szardenings, A. K.; Kozarich, J. W.; Shreder, K. R.
Med. Chem. Lett. 2005, 15, 4239.
13. In our assay, the DPP7 IC50 value for 1-[Dab]-piperidine
was determined to be 69 nM.
14. (a) Senten, K.; Van der Veken, P.; Bal, G.; De Meester, I.;
In conclusion, boro-Nle is a novel P1 residue for the de-
sign of DPP7 selective dipeptide inhibitors. When com-
pared to their boro-Pro congeners, Nle-boro-Nle (4d)
and Dab-boro-Nle (4g) were shown to be significantly
more selective for DPP7. Factors such as the choice of
P2 residue, the presence of the boronic acid, and the
P1 alkyl chain length all play a role in the DPP7 selectiv-
ity and potency of boro-Nle-based inhibitors. The most
DPP7 selective inhibitor found in this study was Dab-
boro-Nle (4g). As such, this compound holds promise
as a tool that can be used to investigate DPP7 biology.
This research sets the stage for the design of other
DPP selective dipeptides inhibitors which incorporate
amino boronic acids based on P1 residues other than
the canonical proline. Reports along these lines will be
presented in due course.
´
Lambeir, A.-M.; Scharpe, S.; Bauvois, B.; Haemers, A.;
Augustyns, K. Bioorg. Med. Chem. Lett. 2002, 12, 2825;
(b) Senten, K.; Van der Veken, P.; De Meester, I.;
´
Lambeir, A. M.; Scharpe, S.; Haemers, A.; Augustyns,
K. J. Med. Chem. 2003, 46, 5005.
15. Senten, K.; Van der Veken, P.; De Meester, I.; Lambeir,
A. M.; Scharpe, S.; Haemers, A.; Augustyns, K. J. Med.
Chem. 2004, 47, 2906.
16. (a) Matteson, D. S.; Jesthi, P. K.; Sadhu, K. M.
Organometallics 1984, 3, 1284; (b) Matteson, D. S. In
Stereodirected Synthesis with Organoboranes; Springer:
Berlin, 1995; (c) Matteson, D. S. Chem. Rev. 1989, 89,
1535.
1
17. Compound 2: H NMR (400 MHz) d (CDCl3): 4.37 (dd,
1H, J = 2.0 Hz, J = 8.8 Hz), 3.47 (dd, 1H, J = 6.4 Hz,
J = 8.4 Hz), 2.30 (m, 2H), 2.09 (t, 1H, J = 5.2 Hz), 7.41 (m,
4H), 1.92 (m, 2H), 1.87 (m, 2H), 1.54 (s, 3H), 1.36 (m, 4H),
1.29 (s, 3H), 1.81 (d, 2H, J = 8.4 Hz), 0.91 (t, 3H
J = 7.2 Hz), 0.85 (s, 3H).
References and notes
1. Rosenblum, J. S.; Kozarich, J. W. Curr. Opin. Chem. Biol.
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2. Underwood, R.; Chiravuri, M.; Lee, H.; Schmitz, T.;
Kabcenell, A. K.; Yardley, K.; Huber, B. T. J. Biol. Chem.
1999, 274, 34053.
3. Chiravuri, M.; Schmitz, T.; Yardley, K.; Underwood, R.;
Dayal, Y.; Huber, B. T. J. Immunol. 1999, 163, 3092.
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A.; Craik, C. S.; Ellman, J. A.; Cummings, R. T.;
Thornberry, N. A. Biochem. J. 2003, 371, 525.
5. Coutts, S. J.; Kelly, T. A.; Snow, R. J.; Kennedy, C. A.;
Barton, R. W.; Adams, J.; Krolikowski, D. A.; Freeman,
D. M.; Campbell, S. J.; Ksiazek, J. F.; Bachovchin, W. W.
J. Med. Chem. 1996, 39, 2087.
18. Coutts, S. J.; Adams, J.; Krolikowski, D.; Snow, R. J.
Tetrahedron Lett. 1994, 35, 5109.
19. Human DPP7, DPP4, and FAP IC50 values were obtained
as described in Ref. 12. Human DPP8 and DPP9 IC50
values were determined via competition between the test
compound and the active site labeling of these enzymes by
FP-TAMRA, a fluorophosphonate-derived, tetramethyl-
rhodamine-tagged activity-based probe for serine hydro-
lases (for more information on this technique, see: Leung,
D.; Hardouin, C.; Boger, D. L.; Cravatt, B. F. Nat.
Biotechnol. 2003, 21, 687, Briefly, MDA-MB-435 cell
lysate was used as a source of DPP8 and DPP9. To
determine IC50 values, test compounds were dissolved in
50% DMSO/50 mM glycine buffer, pH 2.5 (the final
concentration of DMSO in the assays was 1% v/v), then
serially diluted into the lysate to yield; finally, eight
different concentrations ranging from 10 lM to 0.64 nM.
6. (a) Parmee, E. R.; He, J.; Mastracchio, A.; Edmondson, S.
D.; Colwell, L.; Eiermann, G.; Feeney, W. P.; Habulihaz,
B.; He, H.; Kilburn, R.; Leiting, B.; Lyons, K.; Marsilio,
F.; Patel, R. A.; Petrov, A.; Di Salvo, J.; Wu, J. K.;

4260
K. R. Shreder et al. / Bioorg. Med. Chem. Lett. 15 (2005) 4256–4260
FP-TAMRA (1 lM final concentration) was then added.
incubated prior to the inhibition assay. For more infor-
mation, see: Snow, R. J.; Bachovchin, W. W.; Barton, R.
W.; Campbell, S. J.; Coutts, S. J.; Freeman, D. M.;
Gutheil, W. G.; Kelly, T. A.; Kennedy, C. A.; Krolikow-
ski, D. A.; Leonard, S. F.; Pargellis, C. A.; Tong, L.;
Adams, J. J. Am. Chem. Soc. 1994, 116, 10860.
After an incubation time of 30 min the mixture was
quenched with loading buffer. All measurements were
carried out in duplicate. Unreacted probe was separated
from probe-labeled DPP8 or DPP9 using SDS–PAGE and
the fluorescent signal of each TAMRA-tagged enzyme was
measured using a flatbed gel scanner. Samples containing
enzyme alone were considered 100% active and test
compound treated samples were expressed as a percentage
of remaining activity. Percent remaining activity versus
inhibitor concentration was plotted and fit to the Hill
equation to quantify IC50 values.
21. Examples of DPP4 substrates with alanine in the penul-
timate and P1 position include GIP and GLP-1. See:
Mentlein, R.; Gallwitz, B.; Schmidt, W. E. Eur. J.
Biochem. 1993, 214, 829.
22. Interestingly, depending on the P10 residue, alanine can be
an acceptable P1 residue for DPP7 substrates. See: Maes,
M. B.; Lambeir, A. M.; Gilany, K.; Senten, K.; Van der
Veken, P.; Leiting, B.; Augustyns, K.; Scharpe, S.; De
Meester, I. Biochem. J. 2004, 386, 315.
20. Because of the pH-dependent cyclization of boro-Pro
dipeptides via B–N bond formation, IC50 values are
dependent on the pH at which a compound is pre-